High‐Fidelity Trapping of Spatial–Temporal Mitochondria with Rational Design of Aggregation‐Induced Emission Probes

High‐fidelity trapping of mitochondrial dynamic activity is critical to value cellular functions and forecast disease but lack of spatial–temporal probes. Given that commercial mitochondria probes suffering from low photostability, aggregation‐caused quenching effect, and limited signal‐to‐noise ratio from fluorescence “always on” in the process of targeting mitochondria, here, the rational design strategy of a novel aggregation‐induced emission (AIE) molecular motif and unique insight into the high‐fidelity targeting of mitochondria is reported, thereby illustrating the relationship between tailoring molecular aggregation state and mitochondrial targeting ability. This study focuses on how to exactly modulate the hydrophilicity and the aggregated state for realizing “off‐on” fluorescence, as well as matching the charge density to go across the cell membrane for mitochondrial targeting. Probe tricyano‐methylene‐pyridine (TCM‐1) exhibits an unprecedented high‐fidelity feedback on spatial–temporal mitochondrial information with several advantages such as “off‐on” near‐infrared characteristic, high targeting capacity, favorable biocompatibility, as well as excellent photostability. TCM‐1 also produces reactive oxygen species in situ for image‐guided photodynamic anticancer therapy. Through unraveling the relationship between tuning molecular aggregation behavior and organelle‐specific targeting ability, for the first time, a unique guide is provided in designing AIE‐active probes to explore the hydrophilicity and membrane potential for targeting subcellular organelles.     

Red AIE‐Active Fluorescent Probes with Tunable Organelle‐Specific Targeting

Cell staining is a fascinating research area where monitoring and visualizing different cell organelles can be done using fluorescence techniques. However, the design and synthesis of organelle‐targeting fluorophores is still a challenge for several specific organelles. Herein, a platform for synthesizing efficient red‐emitting aggregation‐induced emission luminogens (AIEgens) with donor–acceptor characteristics is reported. The core molecule can be easily functionalized in order to modulate organelle targeting. The three synthesized AIEgens exhibit quantum yields of up to 39.3% and two‐photon absorption cross‐section values of up to 162 GM. The two zwitterionic AIEgens, CDPP‐3SO₃ and CDPP‐4SO_3, with the sulfonate function group, are successfully utilized for specific one‐photon and two‐photon imaging of the endoplasmic reticulum (ER) in live human cells. Substituting the zwitterionic nature with a singly positive charge group, one‐photon and two‐photon imaging of CDPP‐BzBr shows mitochondrial specificity, indicating the importance of the zwitterionic group for ER‐targeting. Owing to the good in vitro photostability, cell viability, and high efficiency, these red dyes serve as a good potential candidate for specific organelle targeting, as well as illustrate how such a platform can easily aid in the study of structure–property relationships for designing such probes.     

Synthesis of Imidazole‐Based AIEgens with Wide Color Tunability and Exploration of their Biological Applications

Research on aggregation‐induced emission (AIE) has become increasingly popular recently and various AIE luminogens (AIEgens) have been developed based on tetraphenylethene, hexaphenylsilole, distyrylanthracene, tetraphenylpyrazine, etc. However, facile tuning of the AIEgen emissions in a wide range remains challenging. Herein, a novel series of AIEgens is reported, based on imidazole‐cored molecular rotors, with facile synthesis and emission colors covering the whole visible spectrum. Moreover, these imidazole derivatives exhibit biological functions unique among the AIEgens, including mitochondria‐specific imaging and antifungal activity. Benefiting from the easy preparation and the tunable emission, the imidazole derivatives are expected to not only diversify the family of AIEgens but also enrich their biological applications.     

Spatiotemporal Theranostic Nanoprobes Dynamically Monitor Targeted Tumor Therapy

Currently, spatiotemporal theranostic nanoprobes are in great demand, owing to their enhanced target therapy and precise dynamic tracing of in vivo drug fate. Herein, this study highlights the successful development of dynamic theranostic nanoprobes, which are facilely established via self-assembly between glutathione (GSH)-responsive dasatinib (DAS) dimers and indocyanine green (ICG). The DAS dimers endow the nanoprobes with aggregation-induced emission (AIE) characteristic, whose emission wavelength successfully redshifts from 420 to 810 nm compared to DAS-based nanoprobes, the same as that of ICG, thus improving the total fluorescence intensity. Moreover, the nanoprobes exhibit a dynamic fluorescence intensity conversion that first decreases and then increases at the tumor site via intracellular GSH-triggered AIE quenching and fluorescence re-enhancement of ICG, therefore achieving precise tumor diagnosis, prognosis evaluation, and spatiotemporal tracing of drug fate compared to other imaging strategies. Furthermore, the nanoprobes show long-term circulation stability via suitable particle sizes and zeta potentials, improved tumor accumulation via extracellular protonation and active cellular uptake, efficient drug release via response to the intracellular milieu, and enhanced apoptosis via targeting to intracellular kinase, therefore achieving the significant tumor inhibition. Thus, the spatiotemporal theranostic nanoprobes can dynamically monitor the targeted tumor therapy, greatly advancing their application in clinics.     

Zwitterionic AIEgens: Rational Molecular Design for NIR-II Fluorescence Imaging-Guided Synergistic Phototherapy

Fluorescence imaging in the second near-infrared region (NIR-II) can penetrate tissue at centimeter depths and obtain high image fidelity. However, facile synthesis of small-molecule fluorescent photosensitizers for efficient NIR-II fluorescence imaging as well as photodynamic and photothermal combinatorial therapies is still a challenging task. Herein, a rational design and facile synthesis protocol are reported for a series of novel NIR-emissive zwitterionic luminogens with aggregation-induced emission (AIE) features for cancer phototheranostics. Consistent with the intrinsic features including long emission wavelength, effective reactive oxygen species generation, and excellent photothermal conversion efficiency (35.76%), in vitro and in vivo evaluation show that one of these presented AIE luminogens provides excellent performance in NIR-II fluorescence imaging-guided synergistic phototherapy against cancer.     

De Novo Design of Aggregation-Induced Emission Luminogen for Three-Photon Fluorescence Imaging of Subcortical Structures Excited at Both NIR-III and NIR-IV Windows

Three-photon fluorescence (3PF) imaging excited at 1700 nm window is an enabling technology for visualizing deep brain structures and dynamics. Recently, the 2200 nm window has emerged as the longest excitation window suitable for deep-brain 3PF imaging. Bright fluorescent probes lay the material basis for deep-brain 3PF imaging. Among various fluorescent probes, aggregation-induced emission luminogens (AIEgens) have great potential in 3PF imaging excited at the 1700 nm window in vivo. However, to the best of knowledge, there is no AIEgens applicable to 3PF imaging excited at both the 1700 and 2200 nm windows. To readily fill this gap, here this study designs and synthesizes a novel AIEgen, namely TPE-DPTT-ICP, which generates bright 3PF signals excited at both 1700 and 2200 nm. The accordingly fabricated TPE-DPTT-ICP nanoparticles (NPs) possess excellent water dispersibility, colloidal stability, biocompatibility, photostability and large 3P action cross section, key to in vivo imaging. In mouse brain in vivo, TPE-DPTT-ICP NPs enable deep-brain 3PF imaging of subcortical structures excited at both the two windows, reaching depths of 1640 and 880 µm below the brain surface, respectively. TPE-DPTT-ICP NPs are thus a versatile material simultaneously catering to the need at two infrared optical windows with deep tissue penetration.     

Designing Squaraine Dyes with Bright Deep-Red Aggregation-Induced Emission for Specific and Ratiometric Fluorescent Detection of Hypochlorite

Development of ratiometric fluorescent hypochlorite probes with strong long wavelength fluorescence in aqueous medium, high resistance to photobleaching, high sensitivity and selectivity, and low biological toxicity remains a challenge. In this work, a molecular design strategy is proposed that can transform the traditional squaraine dyes (SQs) with aggregation-caused quenching character into aggregation-induced emission (AIE)-active luminogens by functionalizing the end-groups with tetraphenylethylene units and further introducing hydrophilic sulfonate group as the side chains. The resulting TPE-SQ5 not only emits strong deep-red fluorescence with a high quantum yield of 11.0% and high photostability, but more encouragingly can serve as a ratiometric fluorescent hypochlorite probe with high selectivity and sensitivity (detection limit: 5.6 nm ), which indeed is the first report for SQs. The detailed sensing mechanism study demonstrates that the oxindole product with sulfonate substitution is responsible for the ratiometric fluorescent response. Furthermore, TPE-SQ5 nanoparticles with high biocompatibility and low cytotoxicity are successfully used for ratiometrically imaging exogenous and endogenous hypochlorite in living cells.     

Facile Synthesis of Red/NIR AIE Luminogens with Simple Structures,Bright Emissions,and High Photostabilities,and Their Applications for Specific Imaging of Lipid Droplets and Image‐Guided Photodynamic Therapy

Red/near‐infrared (NIR) fluorescent molecules with aggregation‐induced emission (AIE) characteristics are of great interest in bioimaging and therapeutic applications. However, their complicated synthetic approaches remain the major barrier to implementing these applications. Herein, a one‐pot synthetic strategy to prepare a series of red/NIR‐emissive AIE luminogens (AIEgens) by fine‐tuning their molecular structures and substituents is reported. The obtained AIEgens possess simple structures, good solubilities, large Stokes shifts, and bright emissions, which enable their applications toward in vitro and in vivo imaging without any pre‐encapsulation or ‐modification steps. Excellent targeting specificities to lipid droplets (LDs), remarkable photostabilities, high brightness, and low working concentrations in cell imaging application make them remarkably impressive and superior to commercially available LD‐specific dyes. Interestingly, these AIEgens can efficiently generate reactive oxygen species upon visible light irradiation, endowing their effective application for photodynamic ablation of cancer cells. This study, thus, not only demonstrates a facile synthesis of red/NIR AIEgens for dual applications in simultaneous imaging and therapy, but also offers an ideal architecture for the construction of AIEgens with long emission wavelengths.     

Molecular Engineering to Boost AIE‐Active Free Radical Photogenerators and Enable High‐Performance Photodynamic Therapy under Hypoxia

The severe hypoxia in solid tumors and the vicious aggregation‐caused fluorescence quenching (ACQ) of conventional photosensitizers (PSs) have limited the application of fluorescence imaging‐guided photodynamic therapy (PDT), although this therapy has obvious advantages in terms of its precise spatial–temporal control and noninvasive character. PSs featuring type I reactive oxygen species (ROS) based on free radicals and novel aggregation‐induced emission (AIE) characteristics (AIE‐PSs) could offer valuable opportunities to resolve the above problems, but molecular engineering methods are rare in previous reports. Herein, a strategy is proposed for generating stronger intramolecular charge transfer in electron‐rich anion‐π⁺ AIE‐active luminogens (AIEgens) to help suppress nonradiative internal conversion and to promote radiative and intersystem crossing to boost free radical generation. Systematic and detailed experimental and theoretical calculations prove the proposal herein: the electron‐donating abilities are enhanced in collaborative donors, and the AIE‐PSs exhibit higher performance in near‐infrared fluorescence imaging‐guided cancer PDT in vitro/vivo. This work serves as an important reference for the design of AIE‐active free radical generators to overcome the ACQ and tumor hypoxia challenges in PDT.     

Versatile Prodrug Nanoparticles for Acid‐Triggered Precise Imaging and Organelle‐Specific Combination Cancer Therapy

Integration of chemotherapy with photodynamic therapy (PDT) has been emerging as a novel strategy for treatment of triple negative breast cancer (TNBC). However, the clinical translation of this approach is hindered by the unwanted dark toxicity due to the “always‐on” model and low tumor specificity of currently approved photosensitizer (PS). Here, the design of a multifunctional prodrug nanoparticle (NP) is described for precise imaging and organelle‐specific combination cancer therapy. The prodrug NP is composed of a newly synthesized oxaliplatin prodrug, hexadecyl‐oxaliplatin‐trimethyleneamine (HOT), an acid‐activatable PS, derivative of Chlorin e6 (AC), and functionalized with a targeting ligand iRGD for tumor homing and penetration. HOT displays much higher antitumor efficiency than oxaliplatin by simultaneously inducing mitochondria depolarizing and DNA cross‐linking. AC is specifically activated in the orthotopic or metastatic TNBC tumor for fluorescence imaging and PDT, while it remains inert in blood circulation to minimize the dark toxicity. Under the guide of acid‐activatable fluorescence imaging, PDT and chemotherapy can be synergistically performed for highly efficient regression of TNBC. Taken together, this versatile prodrug nanoplatform could achieve tumor‐specific imaging and organelle‐specific combination therapy, which can provide an alternative option for cancer theranostic.     

[4]Helicene–Squalene Fluorescent Nanoassemblies for Specific Targeting of Mitochondria in Live‐Cell Imaging

Ester, amide, and directly linked composites of squalene and cationic diaza [4]helicenes 1 are readily prepared. These lipid‐dye constructs 2 , 3 , and 4 give in aqueous media monodispersed spherical nanoassemblies around 100–130 nm in diameter with excellent stability for several months. Racemic and enantiopure nanoassemblies of compound 2 are fully characterized, including by transmission electron microscope and cryogenic transmission electron microscope imaging that did not reveal higher order supramolecular structures. Investigations of their (chir)optical properties show red absorption maxima ≈600 nm and red fluorescence spanning up to the near‐infrared region, with average Stokes shifts of 1350–1550 cm^?1. Live‐cell imaging by confocal microscopy reveals rapid internalization on the minute time scale and organelle‐specific accumulation. Colocalization with MitoTracker in several cancer cell lines demonstrates a specific staining of mitochondria by the [4]helicene–squalene nanoassemblies. To our knowledge, it is the first report of a subcellular targeting by squalene‐based nanoassemblies.     

Molecular engineering of “A-D-D-A” dual-emitting-cores emitters with thermally activated delayed fluorescence and aggregation-induced emission characteristics

Endowing thermally activated delayed fluorescence (TADF) emitter with aggregation-induced emission (AIE) peculiarity is of great significance for realizing more promising commercial applications. Herein, two new dual-emitting-cores emitters with a structure of acceptor-donor-donor-acceptor (A-D-D-A), namely 2DBT-BZ-2Cz and 2DFT-BZ-2Cz, were designed and synthesized to explore their luminescence trait. The emitters, adopting dual carbazole as donor segments and dual phenyl ketone in peripheral skeleton as electron acceptor units, were featured with small singlet (S₁)–triplet (T₁) splitting energy (ΔE_ST) of 0.02 eV and 0.01 eV. The efficient thermally activated delayed fluorescence (TADF) characteristics and aggregation-induced emission property make them suitable for nondoped OLED devices. The solution-processed green OLEDs based on 2DBT-BZ-2Cz demonstrated greater device performance with current efficiency of 20.7 cd A⁻¹ and maximum luminescence of 10,000 cd m⁻². This work thus provides the direction to explore luminogens of dual-emitting-cores with TADF and AIE features as promising candidates in solid state lighting.     

Inorganic–Organic Nanocomposites Based on Aggregation-Induced Emission Luminogens

The emergence of aggregation-induced emission (AIE) has significantly facilitated the development of various fields including chemical sensing, bioimaging, theranostics, and photoelectric devices. Aiming to constructing high-performance versatile materials, integrating AIE luminogens with functionalized supporting agents is an intriguing strategy. Inorganic nanomaterials have attracted significant scientific interest in functionalizing AIE luminogens via combining the advantages of them both. In addition to these intrinsic features of specific inorganic materials, including high stability, superior biocompatibility, and facile modification, the stubborn spatial confinement effect of inorganic structure provides AIE moieties with favorable aggregated conditions. Representative work and some very first attempts of constructing AIE luminogens-based inorganic–organic nanocomposites are presented in this review. The sections are organized according to the dimension of inorganic nanomaterials and focused on their construction, mechanism, and application.     

Real‐Time Imaging of Cell Behaviors in Living Organisms by a Mitochondria‐Targeting AIE Fluorogen

Visualizing behaviors of cell populations within living multicellular organisms in real time is of great value to life science but challenging due to the lack of ideal probes. In this work, a biocompatible fluorogen, azide‐functionalized tetraphenylethene pyridinium (TPE‐PyN₃), is reported for noninvasive imaging and sensing within living systems. TPE‐PyN₃ exhibits unique aggregation‐induced emission (AIE) attributes and high affinity to mitochondria, enabling it to achieve specific mitochondrial imaging and long‐term cellular observing with excellent photostability both in vitro and in vivo. The high membrane penetrability of TPE‐PyN₃ allows all of the cells within the living zebrafish embryos to be morphologically visualized and reconstructed in 3D. Moreover, TPE‐PyN₃ is capable of indicating cell apoptosis because of its sensitivity to the change of mitochondrial membrane potential. The findings presented here provide a simple and noninvasive tool for studying behaviors of cell populations in vivo for the first time by a small‐molecule AIE probe.     

Generic Method of Preparing Multifunctional Fluorescent Nanoparticles Using Flash NanoPrecipitation

There is increased demand for nanoparticles with a high fluorescence yield that have the desired excitation wavelength, surface functionalization, and particle size to act as biological probes. Here, a simple, rapid, and robust method, Flash NanoPrecipitation (FNP), to produce such fluorescent nanoparticles is described. This process involves encapsulation of a hydrophobic fluorophore with an amphiphilic biocompatible diblock copolymer in a kinetically frozen state. FNP is used to produce nanoparticles ranging from 30 to 800 nm with fluorescence emission peaks ranging from, but not limited to, 370 nm to 720 nm. Such fluorescent nanoparticles remain stable in aqueous solutions, and, in contrast to soluble dyes, show no photobleaching. Fluorophores and drugs are incorporated into a single nanoparticle, allowing for simultaneous drug delivery and biological imaging. In addition, functionalization of nanoparticle surfaces with disease‐specific ligands permits precise cell targeting. These features make FNP‐produced fluorescent nanoparticles highly desirable for various biological applications.     

Organic Nanoprobe Cocktails for Multilocal and Multicolor Fluorescence Imaging of Reactive Oxygen Species

Hypochlorite (ClO^?) as a highly reactive oxygen species not only acts as a powerful “guarder” in innate host defense but also regulates inflammation‐related pathological conditions. Despite the availability of fluorescence probes for detection of ClO^? in cells, most of them can only detect ClO^? in single cellular organelle, limiting the capability to fully elucidate the synergistic effect of different organelles on the generation of ClO^?. This study proposes a nanoprobe cocktail approach for multicolor and multiorganelle imaging of ClO^? in cells. Two semiconducting oligomers with different π‐conjugation length are synthesized, both of which contain phenothiazine to specifically react with ClO^? but show different fluorescent color responses. These sensing components are self‐assembled into the nanoprobes with the ability to target cellular lysosome and mitochondria, respectively. The mixture of these nanoprobes forms a nano‐cocktail that allows for simultaneous imaging of elevated level of ClO^? in lysosome and mitochondria according to fluorescence color variations under selective excitation of each nanoprobe. Thus, this study provides a general concept to design probe cocktails for multilocal and multicolor imaging.     

Development of Sulfonamide-Functionalized Charge-Reversal AIE Photosensitizers for Precise Photodynamic Therapy in the Acidic Tumor Microenvironment

Tumor-targeted photodynamic therapy (PDT) is desirable as it can achieve efficient killing of tumor cells with no or less harm to normal cells. Herein, a facile molecular engineering strategy is developed for photosensitizers (PSs) with aggregation induced emission (AIE) characteristics and responsive properties to the acidic tumor microenvironment (TME). By the marriage of pH-sensitive sulfonamide moieties with AIE PSs, two near-infrared AIE luminogens called DBP-SPy and DBP-SPh are designed and synthesized. Both luminogens can form negatively charged nanoaggregates in the aqueous medium at physiological pH. The DBP-SPy nanoaggregates undergo surface charge conversion to become positive at pH close to the signature pH of TME, while DBP-SPh nanoaggregates show no such property. The endowed response to acidic TME enables the enhanced cellular uptake of DBP-SPy at pH = 6.8. By contrast, its cellular uptake is much sacrificed at pH 7.4. As a result, under white light irradiation, DBP-SPy nanoaggregates demonstrate a considerable photodynamic therapeutic effect on cancer cells in vitro and excellent tumor growth inhibition in vivo. Hence, this study not only provides an acidic TME-responsive AIE PS for precise PDT, but also inspires new design strategies for AIE-based theragnostic systems with targeting characteristics.     

Bright and Photostable Organic Fluorescent Dots with Aggregation‐Induced Emission Characteristics for Noninvasive Long‐Term Cell Imaging

Efficient long‐term cell tracing in a noninvasive and real‐time manner is of great importance to understand genesis, development, invasion, and metastasis of cancerous cells. Cell penetrating organic dots with aggregation‐ induced emission (AIE) characteristics are successfully developed as long‐term cell trackers. The AIE dots enjoy the advantages of high emission efficiency, large Stokes shift, good biocompatibility, and high photostability, which ensure their good performance in long‐term non‐invasive in vitro cell tracing. Moreover, it is the first report that AIE dots exhibit certain permeability to cellular nucleus, making them attractive potential candidates for nucleus imaging. The AIE dots display superior performance compared to their counterparts of inorganic quantum dots, opening a new avenue in the development of fluorescent probes for monitoring biological processes.     

Molecular Engineering of NIR-II AIE Luminogen Excited at 1700 nm for Ultradeep Intravital Brain Two-Photon Fluorescence Imaging

The limited tissue penetration depth and spatial resolution are the major bottlenecks for deep-brain imaging. In this study, molecular engineering by tailoring electron donors is conducted to develop for the first time an NIR-II (second near-infrared) emissive fluorescence probe, namely DCTBT, for effective deep-brain two-photon fluorescence imaging. Benefiting from its good biocompatibility, high photostability, bright NIR-II emission as aggregates and large two-photon fluorescence action cross section at the 1700 nm excitation window, DCTBT offers the imaging depths of 2180 and 1135 µm in mouse brain with removed and intact skull, respectively. These results are the record depths for brain imaging, compared to all kinds of fluorescent probes and all modalities of multiphoton microscopy at all demonstrated excitation wavelengths. Moreover, with DCTBT labeling, hemodynamic imaging of blood flow in mouse brain vessels down to a depth of 714 µm with the intact skull is achieved. Multiphoton fluorescence imaging with the NIR-II probe DCTBT excited at the 1700 nm window may readily provide methodology for deep-brain structural and hemodynamic research.     

Efficiency Breakthrough of Fluorescence OLEDs by the Strategic Management of “Hot Excitons” at Highly Lying Excitation Triplet Energy Levels

Aggregation-induced emission (AIE) and hybridized local and charge-transfer (HLCT) materials are two kinds of promising electroluminescence systems for the fabrication of high-efficiency organic light-emitting diodes (OLEDs) by harnessing “hot excitons” at the high-lying triplet exciton states (T_n, n ≥ 2). Nonetheless, the efficiency of the resulting OLEDs did not meet expectations due to the possible loss of T_n→T_n−1. Herein, experimental results and theoretical calculations demonstrate the “hot exciton” process between the high-lying triplet state T₃ and the lowest excited singlet state S₁ in an AIE material 4⁗-(diphenylamino)-2″,5″-diphenyl-[1,1″:4′,1″:4″,1′″:4′″,1⁗-quinquephenyl]-4-carbonitrile (TPB-PAPC) and it is found that the Förster resonance energy transfer (FRET) between two molecules can facilitate the “hot exciton” process and inhibit the T₃→T₂ loss by doping a blue fluorescent emitter in TPB-PAPC. Finally, the doped TPB-PAPC blue OLEDs achieve a maximum external quantum efficiency (EQE_max) of 9.0% with a small efficiency roll-off. Furthermore, doping the blue fluorescent emitter in a HLCT material 2-(4-(10-(3-(9H-carbazol-9-yl)phenyl)anthracen-9-yl)phenyl)-1-phenyl-1H-phenanthro[9,10-d] imidazole (PAC) is used as the emission layer, and the resulting blue OLEDs exhibit an EQE_max of 17.4%, realizing the efficiency breakthrough of blue fluorescence OLEDs. This work establishes a physical insight in the design of high-performance “hot exciton” molecules and the fabrication of high-performance blue fluorescence OLEDs.