Theranostic Gold Nanoclusters for NIR-II Imaging and Photodynamic Therapy

Fluorescence imaging in the second near-infrared window(NIR-II, 1000-1700 nm) has demonstrated tremendous promise for biomedical applications, with its extraordinarily high resolution and deep tissue penetration. Ultrasmall gold nanoclusters(AuNCs) have shown unique features for NIR-II imaging, such as photostability and biocompatibility, as compared to organic NIR-II molecules or other inorganic NIR-II nanoparticles. Here, we report the first-in-class protein-capped ultrasmall AuNCs(BSA-AuNCs, BSA=bovine serum albumin) for simultaneous NIR-II imaging and photodynamic therapy. The BSA-AuNCs show a uniform size, high quantum yield and excellent photostability, display a high accumulation and long retention in 4T1 tumor, and are used for clear imaging of blood vessels and lymph nodes. Moreover, laser irradiation of these AuNCs can rapidly trigger ROS generation, leading to effective inhibition of tumor cell growth in vitro and in vivo. This study demonstrates the feasibility of a protein-capped ultrasmall AuNCs platform for theranostic applications by combining NIR-II imaging and photodynamic cancer therapy.     

Novel CD-MOF NIR-II fluorophores for gastric ulcer imaging

Gastric ulcers are one of the most common stomach diseases that often accompanied by inflammation, congestion, edema, scar tissue formation, and pyloric obstruction. Fiberoptic endoscopy and X-ray analysis of the upper GI tract have become the diagnostic procedure of choice for patients. However, conventional diagnosis technology is either invasive or radioactive. Herein, a novel CD-MOF NIR-II fluorophore (GPs-CH1055) was developed. The relative fluorophore intensity was largely consistent at various media and pH buffers, and it can swell into gel particles in solvents and be completely expelled from the gastrointestinal tract without being assimilated. GPs-CH1055 has been further evaluated in vivo, and exhibited strong retention effect on the gastric ulcer sites, bright NIR-II signals with high spatial and temporal resolution. Therefore, GPs-CH1055 shows great promise for realizing real-time gastric ulcer imaging and diagnosis.     

Fluorination Enhances NIR-II Fluorescence of Polymer Dots for Quantitative Brain Tumor Imaging

Here, we describe a fluorination strategy for semiconducting polymers for the development of highly bright second near-infrared region (NIR-II) probes. Tetrafluorination yielded a fluorescence QY of 3.2 % for the polymer dots (Pdots), over a 3-fold enhancement compared to non-fluorinated counterparts. The fluorescence enhancement was attributable to a nanoscale fluorous effect in the Pdots that maintained the molecular planarity and minimized the structure distortion between the excited state and ground state, thus reducing the nonradiative relaxations. By performing through-skull and through-scalp imaging of the brain vasculature of live mice, we quantitatively analyzed the vascular morphology of transgenic brain tumors in terms of the vessel lengths, vessel branches, and vessel symmetry, which showed statistically significant differences from the wild type animals. The bright NIR-II Pdots obtained through fluorination chemistry provide insightful information for precise diagnosis of the malignancy of the brain tumor.     

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.     

NIR-II Fluorescent Brightness Promoted by “Ring Fusion” for the Detection of Intestinal Inflammation

Fluorophores with emission in the second near-infrared window (NIR-II) have displayed salient advantages for biomedical applications. However, the common strategy of reducing the energy bandgap of fluorophores so as to achieve red-shifted wavelengths always leads to compromised fluorescent brightness. Herein, we propose a molecular design concept of “ring-fusion” to modify the acceptor of AIEgen that can extend the luminous wavelength from NIR-I to NIR-II. The fused-acceptor-containing fluorophore yielded, TTQP, has an enhanced absorption coefficient with a higher brightness in nanoparticle formation compared to its NIR-I emissive counterpart (TTQ-DP) with a non-fused acceptor. Theoretical calculation further confirms that the ring fusion can efficiently promote the rigidity and planarity of the electron-deficient core, leading to a lower reorganization energy and nonradiative decay. The TTQP NPs yielded thus allow sensitive NIR-II fluorescence imaging of vasculature and intestinal inflammation in mice models. Therefore, we anticipate that our work will provide a promising molecular-engineering strategy to enrich the library and broaden the application scope of NIR-II fluorophores.     

An Activatable NIR-II Fluorescent Reporter for In Vivo Imaging of Amyloid-β Plaques

Fluorescence imaging in the second near-infrared (NIR-II) window holds great promise for in vivo visualization of amyloid-β (Aβ) pathology, which can facilitate characterization and deep understanding of Alzheimer's disease (AD); however, it has been rarely exploited. Herein, we report the development of NIR-II fluorescent reporters with a donor-π-acceptor (D-π-A) architecture for specific detection of Aβ plaques in AD-model mice. Among all the designed probes, DMP2 exhibits the highest affinity to Aβ fibrils and can specifically activate its NIR-II fluorescence after binding to Aβ fibrils via suppressed twisted intramolecular charge transfer (TICT) effect. With suitable lipophilicity for ideal blood–brain barrier (BBB) penetrability and deep-tissue penetration of NIR-II fluorescence, DMP2 possesses specific detection of Aβ plaques in in vivo AD-model mice. Thus, this study presents a potential agent for non-invasive imaging of Aβ plaques and deep deciphering of AD progression.     

Fluorescent probe for turn-on sensing of l-cysteine by ensemble of AuNCs and polymer protected AuNPs

A new fluorescent probe based on ensemble of gold nanoclusters (AuNCs) and polymer protected gold nanoparticles (AuNPs) for turn-on sensing of l-cysteine was designed and prepared. The AuNCs were protected by bovine serum albumin and had strong fluorescence. The polymer protected AuNPs were synthesized by a facile in situ strategy at room temperature and could quench the fluorescence of AuNCs due to the Förster resonance energy transfer. Interestingly, it has been observed that the quenched fluorescence of AuNCs was recovered by l-cysteine, which could induce the aggregation of polymer protected AuNPs by sulfur group. Then the prepared fluorescent probe was successfully used for determination of l-Cys in human urines, which would have an evolving aspect and promote the subsequent exploration.     

TADF-based NIR-II semiconducting polymer dots for in vivo 3D bone imaging

Intraoperative fluorescence imaging in the second near-infrared (NIR-II) region heralds a new era in image-guided surgery since the success in the first-in-human liver-tumor surgery guided by NIR-II fluorescence. Limited by the conventional small organic NIR dyes such as FDA-approved indocyanine green with suboptimal NIR-II fluorescence and non-targeting ability, the resulting shallow penetration depth and high false positive diagnostic values have been challenging. Described here is the design of NIR-II emissive semiconducting polymer dots (Pdots) incorporated with thermally activated delayed fluorescence (TADF) moieties to exhibit emission maxima of 1064–1100 nm and fluorescence quantum yields of 0.40–1.58% in aqueous solutions. To further understand how the TADF units affect the molecular packing and the resulting optical properties of Pdots, in-depth and thorough density-functional theory calculations were carried out to better understand the underlying mechanisms. We then applied these Pdots for in vivo 3D bone imaging in mice. This work provides a direction for future designs of NIR-II Pdots and holds promising applications for bone-related diseases.

A series of NIR-II fluorescent TADF-incorporated polymer dots were successfully synthesized. The function of the TADF moiety was fully studied and the bio-applications of these polymer dots including bone imaging were also demonstrated.     

Dual Enhancement of Gold Nanocluster Electrochemiluminescence: Electrocatalytic Excitation and Aggregation‐Induced Emission

Ligand‐protected gold nanoclusters (AuNCs) have emerged as a new class of electrochemiluminescence (ECL) luminophores for their interesting catalytic and emission properties, although their quantum yield (Φ_ECL) in aqueous medium is low with a poor mechanistic understanding of the ECL process. Now it is shown that drying AuNCs on electrodes enabled both enhanced electrochemical excitation by an electrocatalytic effect, and enhanced emission by aggregation‐induced ECL (AIECL) for 6‐aza‐2‐thiothymine (ATT) protected AuNCs with triethylamine (TEA) as a coreactant. The dried ATT‐AuNCs/TEA system resulted in highly stable visual ECL with a Φ_ECL of 78 %, and a similar enhancement was also achieved with methionine‐capped AuNCs. The drying enabled dual‐enhancement mechanism has solved a challenging mechanistic problem for AuNC ECL probes, and can guide further rational design of ECL emitters.     

Dual Enhancement of Gold Nanocluster Electrochemiluminescence: Electrocatalytic Excitation and Aggregation-Induced Emission

Ligand-protected gold nanoclusters (AuNCs) have emerged as a new class of electrochemiluminescence (ECL) luminophores for their interesting catalytic and emission properties, although their quantum yield (Φ_ECL) in aqueous medium is low with a poor mechanistic understanding of the ECL process. Now it is shown that drying AuNCs on electrodes enabled both enhanced electrochemical excitation by an electrocatalytic effect, and enhanced emission by aggregation-induced ECL (AIECL) for 6-aza-2-thiothymine (ATT) protected AuNCs with triethylamine (TEA) as a coreactant. The dried ATT-AuNCs/TEA system resulted in highly stable visual ECL with a Φ_ECL of 78 %, and a similar enhancement was also achieved with methionine-capped AuNCs. The drying enabled dual-enhancement mechanism has solved a challenging mechanistic problem for AuNC ECL probes, and can guide further rational design of ECL emitters.     

A Class of Activatable NIR-II Photoacoustic Dyes for High-Contrast Bioimaging

Photoacoustic (PA) imaging is emerging as one of the important non-invasive imaging techniques in biomedical research. Small molecule- second near-infrared window (NIR-II) PA dyes combined with imaging data can provide comprehensive and in-depth in vivo physiological and pathological information. However, the NIR-II PA dyes usually exhibit “always-on” properties due to the lack of a readily optically tunable group, which hinders the further applications in vivo. Herein, a novel class of dyes GX have been designed and synthesized as an activatable NIR-II PA platform, in which the absorption/emission wavelength of GX-5 extends up to 1082/1360 nm. Importantly, the GX dyes have a strong tissue penetration depth and high-resolution for the mouse vasculature structures in NIR-II PA 3D imaging and high signal-to-noise ratio in NIR-II fluorescence (FL) imaging. Furthermore, to demonstrate the applicability of GX dyes, the first NIR-II PA probe GX-5-CO activated by carbon monoxide (CO) was engineered and employed to reveal the enhancement of the CO levels in the hypertensive mice by high-contrast NIR-II PA and FL imaging. We expect that many derivatives of GX dyes will be developed to afford versatile NIR-II PA platforms for designing a wide variety activatable NIR-II PA probes as biomedical tools.     

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...     

Electron-Withdrawing Substituents Allow Boosted NIR-II Fluorescence in J-Type Aggregates for Bioimaging and Information Encryption

Developing molecular fluorophores with enhanced fluorescence in aggregate state for the second near-infrared (NIR-II) imaging is highly desirable but remains a tremendous challenge due to the lack of reliable design guidelines. Herein, we report an aromatic substituent strategy to construct highly bright NIR-II J-aggregates. Introduction of electron-withdrawing substituents at 3,5-aryl and meso positions of classic boron dipyrromethene (BODIPY) skeleton can promote slip-stacked J-type arrangement and further boost NIR-II fluorescence of J-aggregates via increased electrostatic repulsion and intermolecular hydrogen bond interaction. Notably, NOBDP-NO₂ with three nitro groups (−NO₂) shows intense NIR-II fluorescence at 1065 nm and high absolute quantum yield of 3.21 % in solid state, which can be successfully applied in bioimaging, high-level encoding encryption, and information storage. Moreover, guided by this electron-withdrawing substituent strategy, other skeletons (thieno-fused BODIPY, aza-BODIPY, and heptamethine cyanine) modified with −NO₂ are converted into J-type aggregates with enhanced NIR-II fluorescence, showing great potential to convert aggregation caused emission quenching (ACQ) dyes into brilliant J-aggregates. This study provides a universal method for construction of strong NIR-II emissive J-aggregates by rationally manipulating molecular packing and establishing relationships among molecular structures, intermolecular interactions, and fluorescence properties.     

Nonabsorbable Iron(III) binding polymers: Synthesis and evaluation of the chelating properties

Iron is a key micronutrient essential for many biological events. While iron deficiency can lead to anemia, supplementation with oral iron often ends up with enteral iron overload, a critical gastrointestinal (GI) burden linked to the increased risk of dysbiosis, infections and often associated with colorectal cancer. Iron chelation therapy is clinically used to reduce pathological systemic iron overload by established low molecular weight iron chelators. As drawbacks, these drugs present low pharmacokinetic profiles and several toxicities, leading to relatively high rates of adverse effects. To overcome these issues, the prevention of iron accumulation in the GI tract by non-absorbable iron binding polymers could represent an alternative still underexploited approach. Here, we present the development of a series of insoluble polymeric Fe(III) chelators. These innovative compounds have been obtained by the conjugation of 3-hydroxypyridin-4-one Fe(III) chelating moiety with branched Polyethyleneimine (PEI) and Carboxymethyl cellulose (CMC). In vitro binding studies indicated that the Fe(III) chelating capacity depends on the nature of the polymer. In particular, PEI derivatives possess higher selectivity toward Fe(III) in simulated intestinal fluid preserving the integrity of intestinal enterocytes, representing thus promising compounds in the development of iron chelators.     

Selective Killing of Breast Cancer Cells by Doxorubicin‐Loaded Fluorescent Gold Nanoclusters: Confocal Microscopy and FRET

Fluorescent gold nanoclusters (AuNCs) capped with lysozymes are used to deliver the anticancer drug doxorubicin to cancer and noncancer cells. Doxorubicin‐loaded AuNCs cause the highly selective and efficient killing (90 %) of breast cancer cells (MCF7) (IC₅₀=155 nm ). In contrast, the killing of the noncancer breast cells (MCF10A) by doxorubicin‐loaded AuNCs is only 40 % (IC₅₀=4500 nm ). By using a confocal microscope, the fluorescence spectrum and decay of the AuNCs were recorded inside the cell. The fluorescence maxima (at ≈490–515 nm) and lifetime (≈2 ns), of the AuNCs inside the cells correspond to Au_10–13. The intracellular release of doxorubicin from AuNCs is monitored by Förster resonance energy transfer (FRET) imaging.     

NIR-II Chemiluminescence Molecular Sensor for In Vivo High-Contrast Inflammation Imaging

Chemiluminescence (CL) sensing without external excitation by light and autofluorescence interference has been applied to high-contrast in vitro immunoassays and in vivo inflammation and tumor microenvironment detection. However, conventional CL sensing usually operates in the range of 400–850 nm, which limits the performance of in vivo imaging due to serious light scattering effects and signal attenuation in tissue. To address this challenge, a new type of CL sensor is presented that functions in the second near-infrared window (NIR-II CLS) with a deep penetration depth (≈8 mm). Successive CL resonance energy transfer (CRET) and Förster resonance energy transfer (FRET) from the activated CL substrate to two rationally designed donor-acceptor-donor fluorophores BTD540 and BBTD700 occurs. NIR-II CLS can be selectively activated by hydrogen peroxide over other reactive oxygen species (ROSs). Moreover, NIR-II CLS is capable of detecting local inflammation in mice with a 4.5-fold higher signal-to-noise ratio (SNR) than that under the NIR-II fluorescence modality.     

Pre‐oxidation of Gold Nanoclusters Results in a 66 % Anodic Electrochemiluminescence Yield and Drives Mechanistic Insights

Gold nanoclusters (AuNCs) are attractive electrochemiluminescence (ECL) emitters because of their excellent stability, near IR emission, and biocompatibility. However, their ECL quantum yield is relatively low, and our limited fundamental understanding has hindered rational improvement of this parameter. Herein, we report drastic enhancement of the ECL of ligand‐stabilized AuNCs by on‐electrode pre‐oxidation with triethylamine (TEA) as a co‐reactant. The l ‐methionine‐stabilized AuNCs resulted in a record high ECL yield of 66 %. This strategy was successfully extended to other AuNCs, and it is more effective for ligand shells that allow more effective electron transfer. In addition, excitation of the pre‐oxidized ECL required a lower potential than conventional methods, and no additional instrument was required. This work opens avenues for solving a challenging problem of AuNC‐based ECL probes and enriches fundamental understanding, greatly broadening their potential applications.     

Organoboron luminophores with extremely strong dual–phase emissions

Developing efficient dual–phase emission emitters upon organoboron luminophores remains a formidable challenge due to the ubiquitous self–absorption and deleterious π-π interactions from aromatic structure. Here, a new family of benzothiazole–enolate–based organoboron luminophores (HN1–4) with effective dual–phase emission was constructed. HN4 showed almost the highest quantum yield (QY) among this type of compound so far. The three-ring–fused rigid skeleton and moderate intramolecular charge transfer (ICT) effect ensured that HN4 could give rise to extremely strong emission in any solution (QY up to 99%). X-ray crystallographic analysis showed that the twisted core structure constructed by the boronic coordination of two penta-fluorobenzene of HN4 was responsible for intense emission in the solid state (QY up to 68%). Besides, HN4 exhibited a unique response to mechanical force accompanied by a reversible change of the QY. We believe that this strategy provides beneficial inspiration and methodology to design materials with high emissive quantum yield that can be used in a variety of luminescent events.     

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.     

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.