Expanded porphyrins: functional photoacoustic imaging agents that operate in the NIR-II region

Photoacoustic imaging (PAI) relies on the use of contrast agents with high molar absorptivity in the NIR-I/NIR-II region. Expanded porphyrins, synthetic analogues of natural tetrapyrrolic pigments (e.g. heme and chlorophyll), constitute as potentially attractive platforms due to their NIR-II absorptivity and their ability to respond to stimuli. Here, we evaluate two expanded porphyrins, naphthorosarin (1) and octaphyrin (4), as stimuli responsive PA contrast agents for functional PAI. Both undergo proton-coupled electron transfer to produce species that absorb well in the NIR-II region. Octaphyrin (4) was successfully encapsulated into 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG₂₀₀₀) nanoparticles to afford OctaNPs. In combination with PAI, OctaNPs allowed changes in the acidic environment of the stomach to be visualized and cancerous versus healthy tissues to be discriminated.

In this study, two expanded porphyrins, octaphyrin and naphthorosarin were evaluated as potential PA agents. The nanoparticle encapsulation of octaphyrin successfully enabled the visualization of acidic environments and the discrimination between cancerous and healthy tissues.     

NIR-Ⅱ Excitation and NIR-I Emission Based Two-photon Fluorescence Lifetime Microscopic Imaging Using Aggregation-induced Emission Dots

Near-infrared(NIR)lights are powerful tools to conduct deep-tissue imaging since NIR-Ⅰ wavelengths hold less photon absorption and NIR-Ⅱ wavelengths serve low photon scattering in the biological tissues compared with visible lights.Two-photon fluorescence lifetime microscopy(2PFLM)can utilize NIR-Ⅱ excitation and NIR-Ⅰ emission at the same time with the assistance of a well-designed fluorescent agent.Aggregation induced emission(AIE)dyes are famous for unique optical properties and could serve a large two-photon absorption(2PA)cross-section as aggregated dots.Herein,we report two-photon fluorescence lifetime microscopic imaging with NIR-Ⅱ excitation and NIR-Ⅰ emission using a novel deep-red AIE dye.The AIE-gens held a 2PA cross-section as large as 1.61×10⁴GM at 1040 nm.Prepared AIE dots had a two-photon fluorescence peak at 790 nm and a stable lifetime of 2.2 ns under the excitation of 1040 nm femtosecond laser.The brain vessels of a living mouse were vividly reconstructed with the two-photon fluorescence lifetime information obtained by our home-made 2PFLM system.Abundant vessels as small as 3.17μm were still observed with a nice signal-background ratio at the depth of 750μm.Our work will inspire more insight into the improvement of the working wavelength of fluorescent agents and traditional 2PFLM.     

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.     

Structural and process controls of AIEgens for NIR-II theranostics

Aggregation-induced emission (AIE) is a cutting-edge fluorescence technology, giving highly-efficient solid-state photoluminescence. Particularly, AIE luminogens (AIEgens) with emission in the range of second near-infrared window (NIR-II, 1000–1700 nm) have displayed salient advantages for biomedical imaging and therapy. However, the molecular design strategy and underlying mechanism for regulating the balance between fluorescence (radiative pathway) and photothermal effect (non-radiative pathway) in these narrow bandgap materials remain obscure. In this review, we outline the latest achievements in the molecular guidelines and photophysical process control for developing highly efficient NIR-II emitters or photothermal agents with aggregation-induced emission (AIE) attributes. We provide insights to optimize fluorescence efficiency by regulating multi-hierarchical structures from single molecules (flexibilization) to molecular aggregates (rigidification). We also discuss the crucial role of intramolecular motions in molecular aggregates for balancing the functions of fluorescence imaging and photothermal therapy. The superiority of the NIR-II region is demonstrated by fluorescence/photoacoustic imaging of blood vessels and the brain as well as photothermal ablation of the tumor. Finally, a summary of the challenges and perspectives of NIR-II AIEgens for in vivo theranostics is given.

Structural and process controls of NIR-II AIEgens realize manipulating of radiative (R) and nonradiative (NR) decay for precise theranostics.     

Biocompatible organic dots with aggregation-induced emission for in vitro and in vivo fluorescence imaging

Fluorescent probes play a key role in modern biomedical research. As compared to inorganic quantum dots (QDs) composed with heavy metal elements, organic dye-based fluorescent nanoparticles have higher biocompatibility and are richer in variety. However, traditional organic fluorophores tend to quench fluorescence upon aggregation, which is known as aggregation-caused quenching (ACQ) effect that hinders the fabrication of highly emissive fluorescent nanoparticles. In this work, we demonstrate the synthesis of organic fluorescent dots with aggregation-induced emission (AIE) in far-red/near-infrared (FA/NIR) region. A conventional ACQ-characteristic fluorescent dye, 3,4:9,10-tetracarboxylic perylene bisimide (PBI), is converted into an AIE fluorogen through attaching two tetraphenylethylene (TPE) moieties. The fluorescent dots with surface folic acid groups are fabricated from PBI derivative (DTPEPBI), showing specific targeting effect to folate receptor-overexpressed cancer cells. In vivo studies also suggest that the folic acid-functionalized AIE dots preferentially accumulate in the tumor site through enhanced permeability and retention (EPR) effect and folate receptor-mediated active targeting effect. The low cyto-toxicity, good FR/NIR contrast and excellent targeting ability in in vitro/in vivo imaging indicate that the AIE dots have great potentials in advanced bioimaging applications.     

Sub-10 nm Aggregation-Induced Emission Quantum Dots Assembled by Microfluidics for Enhanced Tumor Targeting and Reduced Retention in the Liver

A robust platform is developed to assemble sub-10 nm organic aggregation-induced emission (AIE) particles using four different AIE luminogens (AIEgens) with emissions from green to the second near-infrared window (NIR-II). They are called AIE quantum dots (QDs) to distinguish from typical AIE dots which are larger than 25 nm. Compared with AIE dots that are larger than 25 nm, AIE QDs allow more efficient cellular uptake and imaging without surface modification of any membrane-penetrating peptides or other targeting molecules. NIR-II AIEgens, which have nearly no background fluorescence from organisms, are used to demonstrate that AIE QDs can achieve high contrast at the tumor as small as 80 mm³ and evade the liver more efficiently than AIE dots. AIE QDs hold a good promise for sensitive and precise diagnosis of the latent solid tumor in clinical medicine with much lower off-targeting to the liver than AIE dots.     

Near-infrared fluorescent molecular probes for imaging and diagnosis of nephro-urological diseases

Near-infrared (NIR) fluorescence imaging has improved imaging depth relative to conventional fluorescence imaging in the visible region, demonstrating great potential in both fundamental biomedical research and clinical practice. To improve the detection specificity, NIR fluorescence imaging probes have been under extensive development. This review summarizes the particular application of optical imaging probes with the NIR-I window (700–900 nm) or the NIR-II window (1000–1700 nm) emission for diagnosis of nephron-urological diseases. These molecular probes have enabled contrast-enhanced imaging of anatomical structures and physiological function as well as molecular imaging and early diagnosis of acute kidney injury, iatrogenic ureteral injury and bladder cancer. The design strategies of molecular probes are specifically elaborated along with representative imaging applications. The potential challenges and perspectives in this field are also discussed.

Near-infrared fluorescent molecular probes with improved imaging depth and optimized biodistribution have been reviewed, showing great potential for diagnosis of nephro-urological diseases.     

Rational design of an “all-in-one” phototheranostic

We report here porphodilactol derivatives and their corresponding metal complexes. These systems show promise as “all-in-one” phototheranostics and are predicated on a design strategy that involves controlling the relationship between intersystem crossing (ISC) and photothermal conversion efficiency following photoexcitation. The requisite balance was achieved by tuning the aromaticity of these porphyrinoid derivatives and forming complexes with one of two lanthanide cations, namely Gd³⁺ and Lu³⁺. The net result led to a metalloporphodilactol system, Gd-trans-2, with seemingly optimal ISC efficiency, photothermal conversion efficiency and fluorescence properties, as well as good chemical stability. Encapsulation of Gd-trans-2 within mesoporous silica nanoparticles (MSN) allowed its evaluation for tumour diagnosis and therapy. It was found to be effective as an “all-in-one” phototheranostic that allowed for NIR fluorescence/photoacoustic dual-modal imaging while providing an excellent combined PTT/PDT therapeutic efficacy in vitro and in vivo in 4T1-tumour-bearing mice.

We report here porphodilactol derivatives and their corresponding metal complexes as “all-in-one” phototheranostics by controlling the relationship between intersystem crossing (ISC) and photothermal conversion efficiency following photoexcitation.     

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.     

Facilely prepared aggregation-induced emission (AIE) nanocrystals with deep-red emission for super-resolution imaging

Organic nanocrystals (NCs) with high brightness are highly desirable for biological imaging. However, the preparation of NCs by a facile and fast method is still challenging. Herein, an aggregation-induced emission (AIE) luminogen of 4,4′-(5,6-difluorobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(N,N-bis(4-methoxyphenyl)aniline) (DTPA-BT-F) in the deep-red region is designed with intensive crystalline features to obtain NCs by kinetically controlled nanoprecipitation. The prepared AIE NCs with high brightness and good photo-stability are then applied in super-resolution imaging via stimulated emission depletion (STED) nanoscopy. As observed, the nanostructures in lysosomes of both fixed and live cells are well visualized with superior lateral resolutions under STED nanoscopy (full width at half maximum values, 107 and 108 nm) in contrast to that in confocal imaging (548 and 740 nm). More importantly, dynamic monitoring and long-term tracking of lysosomal movements in live HeLa cells, such as lysosomal contact, can also be carried out by using DTPA-BT-F NCs at a superior resolution. To the best of our knowledge, this is the first case of AIE NCs prepared by nanoprecipitation for STED nanoscopy, thus providing a new strategy to develop high performance imaging agents for super-resolution imaging.

AIE nanocrystals with high brightness in the deep-red region were facilely prepared by kinetically controlled nanoprecipitation. These nanocrystals were then applied in super-resolution cellular imaging via STED nanoscopy.     

Recent advances of AIE light-up probes for photodynamic therapy

As a new non-invasive treatment method, photodynamic therapy (PDT) has attracted great attention in biomedical applications. The advantages of possessing fluorescence for photosensitizers have made it possible to combine imaging and diagnosis together with PDT. The unique features of aggregation-induced emission (AIE) fluorogens provide new opportunities for facile design of light-up probes with high signal-to-noise ratios and improved theranostic accuracy and efficacy for image-guided PDT. In this review, we summarize the recent advances of AIE light-up probes for PDT. The strategies and principles to design AIE photosensitizers and light-up probes are firstly introduced. The application of AIE light-up probes in photodynamic antitumor and antibacterial applications is further elaborated in detail, from binding/targeting-mediated, reaction-mediated, and external stimuli-mediated light-up aspects. The challenges and future perspectives of AIE light-up probes in the PDT field are also presented with the hope to encourage more promising developments of AIE materials for phototheranostic applications and translational research.

AIE fluorogens provide new opportunities for the development of light-up probes for photodynamic therapy.     

Probing biotin receptors in cancer cells with rationally designed fluorogenic squaraine dimers

Fluorogenic probes enable imaging biomolecular targets with high sensitivity and maximal signal-to-background ratio under non-wash conditions. Here, we focus on the molecular design of biotinylated dimeric squaraines that undergo aggregation-caused quenching in aqueous media through intramolecular H-type dimerization, but turn on their fluorescence in apolar environment due to target-mediated disaggregation. Our structure–property study revealed that depending on the linkers used to connect the squaraine dyes, different aggregation patterns could be obtained (intramolecular dimerization versus intermolecular aggregation) leading to different probing efficiencies. Using a relatively short l-lysine linker we developed a bright fluorogenic probe, Sq2B, displaying only intramolecular dimerization-caused quenching properties in aqueous media. The latter was successfully applied for imaging biotin receptors, in particular sodium-dependent multivitamin transporter (SMVT), which are overexpressed at the surface of cancer cells. Competitive displacement with SMVT-targets, such as biotin, lipoic acid or sodium pantothenate, showed Sq2B targeting ability to SMVT. This fluorogenic probe for biotin receptors could distinguish cancer cells (HeLa and KB) from model non-cancer cell lines (NIH/3T3 and HEK293T). The obtained results provide guidelines for development of new dimerization-based fluorogenic probes and propose bright tools for imaging biotin receptors, which is particularly important for specific detection of cancer cells.

Rational design of self-quenched squaraine dimers bearing biotin yielded a bright fluorogenic probe that can distinguish cancerous from non-cancerous cells.     

Fluorescent AIE dots encapsulated organically modified silica (ORMOSIL) nanoparticles for two-photon cellular imaging

2,3-Bis(4-(phenyl(4-(1,2,2-triphenylvinyl)phenyl)amino)phenyl) fumaronitrile (TPE-TPA-FN or TTF), which possesses aggregation-induced emission (AIE) characteristic, is doped in organically modified silica (ORMOSIL) nanoparticles. By increasing the weight ratio of TTF to the precursor of silica nanoparticles (the quantities of the precursors were kept the same), the fluorescence intensity of nanoparticles increased correspondingly, due to the formation of larger AIE dots in the cores of ORMOSIL nanoparticles. The fluorescent and biocompatible nanoprobes were then utilized for in vitro imaging of HeLa cells. Two-photon fluorescence microscopy clearly illustrated that the nanoparticles have the capacity of nucleus permeability, as well as cytoplasm staining towards tumor cells. Our experimental results may offer a promising method for fast and bright fluorescence imaging, as well as bio-molecule/drug delivery to cell nucleus.     

Boron difluoride formazanate dye for high-efficiency NIR-Ⅱ fluorescence imaging-guided cancer photothermal therapy

The small molecular second near-infrared(NIR-Ⅱ, 1000–1700 nm) dye-based nanotheranostics can concurrently combine deep-tissue photodiagnosis with in situ phototherapy, which occupies a vital position in the early detection and precise treatment of tumors. However, the development of small molecular NIR-Ⅱ dyes is still challenging due to the limited electron acceptors and cumbersome synthetic routes.Herein, we report a novel molecular electron acceptor, boron difluoride formazanate(BDF). Based on...     

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).     

Aggregation-induced delayed fluorescence luminogens: the innovation of purely organic emitters for aqueous electrochemiluminescence

Due to overcoming the limitation of aggregation caused quenching (ACQ) of solid-state emitters, aggregation-induced emission (AIE) organic luminogens have become a promising candidate in aqueous electrochemiluminescence (ECL). However, restricted by the physical nature of fluorescence, current organic AIE luminogen-based ECL (AIECL) faces the bottleneck of low ECL efficiency. Here, we propose to construct de novo aqueous ECL based on aggregation-induced delayed fluorescence (AIDF) luminogens, called AIDF-ECL. Compared with the previous organic AIE luminogens, purely organic AIDF luminogens integrate the superiorities of both AIE and the utilization of dark triplets via thermal-activated spin up-conversion properties, thereby possessing the capability of close-to-unity exciton utilization for ECL. The results show that the ECL characteristics using AIDF luminogens are directly related to their AIDF properties. Compared with an AIECL control sample based on a tetraphenylethylene AIE moiety, the ECL efficiency of our AIDF-ECL model system is improved by 5.4 times, confirming the excellent effectiveness of this innovative strategy.

Electrochemiluminescence (ECL) using an aggregation-induced delayed fluorescence (AIDF) organic luminogen, i.e. AIDF-ECL, was reported for the first time, featuring “lighting” dark triplets (ca. 75% in total) for all-exciton-harvesting ECL applications.     

Simultaneously achieving high capacity storage and multilevel anti-counterfeiting using electrochromic and electrofluorochromic dual-functional AIE polymers

With the advent of the big data era, information storage and security are becoming increasingly important. However, high capacity information storage and multilevel anti-counterfeiting are typically difficult to achieve simultaneously. To address this challenge, herein, two electrochromic and electrofluorochromic dual-functional polymers with aggregation-induced emission (AIE) characteristics were rationally designed and facilely prepared. Upon applying voltages, the absorption and fluorescence spectra of the AIE polymers can undergo reversible changes, accompanied by variation of their color and emission. By utilizing the controllable characteristics of the polymers, dual-mode display devices were fabricated via a simple spraying technique. More interestingly, a four-dimensional color code device was constructed by adding color change multiplexing to the two-dimensional space, thereby achieving high capacity information storage. Moreover, the color code device can also be applied in the multilevel anti-counterfeiting area. The encrypted information can be dynamically converted under different voltages. Thus, the AIE polymers show great promise for applications in multidimensional information storage and dynamic anti-counterfeiting, and the design strategy may provide a new avenue for advanced information storage and high security technology.

By using electrical stimuli-responsive AIE polymers, dual-mode display devices, multidimensional information storage and anti-counterfeiting devices were constructed.     

Fluorescent polymer cubosomes and hexosomes with aggregation-induced emission

Fluorescent polymer cubosomes and hexosomes with aggregation-induced emission (AIE) were prepared from amphiphilic block copolymers PEG-b-PTPEMA where the hydrophobic block PTPEMA was a polymethacrylate with tetraphenylethene (TPE) as the AIE side group. Four highly asymmetric block copolymers with hydrophilic block weight ratio f_PEG ≤ 20% were synthesized. Cubosomes and hexosomes with strong fluorescence emission were obtained by nanoprecipitation of polymers with f_PEG < 9% in dioxane/water and THF/water systems. Their ordered internal structures were studied by electron microscopy (cryo-EM, SEM and TEM) and the X-ray scattering technique (SAXS). To elucidate the formation mechanisms of these inverted colloids, other parameters influencing the morphologies, like the water content during self-assembly and the organic solvent composition, were also investigated. This study not only inspires people to design novel building blocks for the preparation of functional cubosomes and hexosomes, but also presents the first AIE fluorescent polymer cubosome and hexosome with potential applications in bio-related fields.

Fluorescent Im$\bar{3}$m cubosome and P6mm hexosome with aggregation-induced emission (AIE) were reported, which were formed by amphiphilic block copolymers PEG-b-PTPEMA. The length of hydrophobic block PTPEMA was adjusted to control morphology formation.     

AIE-based nanoaggregate tracker: high-fidelity visualization of lysosomal movement and drug-escaping processes

High-fidelity imaging and long-term visualization of lysosomes are crucial for their functional evaluation, related disease detection and active drug screening. However, commercial aggregation-caused quenching probes are not conducive to precise lysosomal imaging because of their inherent drawbacks, like easy diffusion, short emission and small Stokes shift, let alone their long-term tracing due to rapid photobleaching. Herein we report a novel aggregation-induced emission (AIE)-based TCM-PI nanoaggregate tracker for direct visualization of lysosomes based on the building block of tricyano-methylene-pyridine (TCM), wherein introduced piperazine (PI) groups behave as targeting units to lysosomes upon protonation, and the self-assembled nanostructure contributes to fast endocytosis for enhanced targeting ability as well as extended retention time for long-term imaging. The piperazine-stabilized TCM-PI nanoaggregate shifts the emission maximum to 677 nm in an aqueous environment, and falls within the desirable NIR region with a large Stokes shift of 162 nm, thereby greatly reducing biological fluorescent background interference. In contrast with the commercially available LysoTracker Red, the essential AIE characteristic of high photostability can guarantee three-dimensional high-fidelity tracing with low photobleaching, and little diffusion from lysosomes, and especially overcome the AIE bottleneck to target specificity. Consequently, the AIE-based nanoaggregate tracker successfully achieves the high-fidelity and long-term tracing of lysosomal movement and even monitors the drug-escaping process from lysosomes to cell nuclei, which provides a potential tool to benefit drug screening.

Well-formed AIE nanoaggregates with good stability can achieve high-fidelity visualization of lysosomal movement and drug-escaping processes.