An Activatable AIEgen Probe for High‐Fidelity Monitoring of Overexpressed Tumor Enzyme Activity and Its Application to Surgical Tumor Excision

Monitoring fluctuations in enzyme overexpression facilitates early tumor detection and excision. An AIEgen probe (DQM‐ALP) for the imaging of alkaline phosphatase (ALP) activity was synthesized. The probe consists of a quinoline‐malononitrile (QM) core decorated with hydrophilic phosphate groups as ALP‐recognition units. The rapid liberation of DQM‐OH aggregates in the presence of ALP resulted in aggregation‐induced fluorescence. The up‐regulation of ALP expression in tumor cells was imaged using DQM‐ALP. The probe permeated into 3D cervical and liver tumor spheroids for imaging spatially heterogeneous ALP activity with high spatial resolution on a two‐photon microscopy platform, providing the fluorescence‐guided recognition of sub‐millimeter tumorigenesis. DQM‐ALP enabled differentiation between tumor and normal tissue ex vivo and in vivo, suggesting that the probe may serve as a powerful tool to assist surgeons during tumor resection.     

用于肿瘤成像的半乳糖/酞菁近红外荧光探针

本文首次报道半乳糖酞菁近红外荧光探针在肿瘤成像方面的应用。以酞菁为荧光发射基团,其在近红外区域有较高的量子产量和光学稳定性,可以解决探针的近红外光学特性;通过半乳糖对酞菁的修饰能够改善探针的溶解性和生物相容性,而且利用半乳糖的肿瘤靶向功能可以提高探针肿瘤主动靶向成像效果。结果表明它对恶性肿瘤具有高特异性结合和高灵敏度的分子探针体系,提高近红外光学分子成像效果。     

A Light‐Up Probe with Aggregation‐Induced Emission for Real‐Time Bio‐orthogonal Tumor Labeling and Image‐Guided Photodynamic Therapy

Bio‐orthogonal tumor labeling is more effective in delivering imaging agents or drugs to a tumor site than active targeting strategy owing to covalent ligation. However, to date, tumor‐specific imaging through bio‐orthogonal labeling largely relies on body clearance to differentiate target from the intrinsic probe signal owing to the lack of light‐up probes for in vivo bio‐orthogonal labeling. Now the first light‐up probe based on a fluorogen with aggregation‐induced emission for in vivo bio‐orthogonal fluorescence turn‐on tumor labeling is presented. The probe has low background fluorescence in aqueous media, showing negligible non‐specific interaction with normal tissues. Once it reacts with azide groups introduced to tumor cells through metabolic engineering, the probe fluorescence is lightened up very quickly, enabling rapid tumor‐specific imaging. The photosensitizing ability was also used to realize effective image‐guided photodynamic tumor therapy.     

A Fluorescent Probe with Aggregation‐Induced Emission for Detecting Alkaline Phosphatase and Cell Imaging

Alkaline phosphatase (ALP) is associated with many diseases, and its accurate detection is of great significance. Fluorescent compounds with aggregation‐induced emission (AIE) feature show beneficial advantages for serving as fluorescent probes. Herein, an AIE‐active “turn on” probe for ALP detection was synthesized through incorporating a strong electron‐withdrawing group (cyano) in the middle and the recognition moiety phosphate group at the end, thereby rendering a D–A–D structure with a relatively high conjugation degree and good water solubility. It was found that the probe TPE‐CN‐pho is highly sensitive to ALP in aqueous solution. In the presence of ALP, the hydrophilic phosphate group on the probe is rapidly removed, resulting in a decrease in water solubility and subsequent formation of aggregates, thereby achieving aggregation‐induced emission. Moreover, the probe TPE‐CN‐pho has also been successfully applied to imaging ALP in living cells.     

Activatable nanoscale metal-organic framework for ratiometric photoacoustic imaging of hydrogen sulfide and orthotopic colorectal cancer in vivo

Nanoscale metal-organic frameworks(nano MOFs) have emerged as a promising biomedical nanoplatform because of their unique properties. However, the exploration of nano MOFs in photoacoustic(PA) imaging is still limited. Here, a novel hydrogen sulfide(H2 S)-activated nano copper-based MOF(Cu-MOF) was developed as a near-infrared(NIR) ratiometric PA probe for in vivo monitoring of endogenous H2 S level and orthotopic colorectal cancer imaging via in situ reaction of nano Cu-MOFs with endogenous H_2 S that is closely associated with tumor growth and proliferation in colon cancer. The synthesized nano Cu-MOFs displayed excellent PA responsiveness towards tumor H_2 S level with high selectivity and rapid kinetics. The result suggests the developed probe may provide a unique opportunity to investigate the malignant behaviors of H_2 S-associated events in vivo.     

Optimized Fluorescent Probe for Specific Imaging of Glutathione S‐Transferases in Living Cells and Mice

GSTP1 has been considered to be a marker for malignancy in many tissues. However, the existing GST fluorescent probes are unfavorable for in vivo imaging because of the limited emission wavelength or insufficient fluorescence enhancement (six‐fold). The limited fluorescence enhancement of GST fluorescent probes is mainly ascribed to the high background signals resulting from the spontaneous reaction between GSH and the probes. In this work, a highly specific GST probe with NIR emission has been successfully developed through optimization of the essential unit of the probe to repress the spontaneous reaction. The novel GST probe exhibits over 100‐fold fluorescence enhancement upon incubation with GSTP1/GSH and high selectivity over other potential interference. In addition, the probe has been proved to be capable of tracking endogenous GST in A549 cells. Finally, the in vivo imaging results demonstrate that the probe can be used for effective imaging of endogenous GST activity in subcutaneous tumor mouse with high contrast.     

A fluorescent turn-on probe for the detection of alkaline phosphatase activity in living cells

Fluorescent probe 1, showing a high fluorescence turn-on signal ratio, enables the real-time imaging of endogenous alkaline phosphatase (ALP) activity in living cells, and the fast and quantitative analysis of enzyme activity at the single-cell level.     

An enzymatically activated fluorescence probe for targeted tumor imaging

Beta-galactosidase is a widely used reporter enzyme, but although several substrates are available for in vitro detection, its application for in vivo optical imaging remains a challenge. To obtain a probe suitable for in vivo use, we modified our previously developed activatable fluorescence probe, TG-betaGal (J. Am. Chem. Soc. 2005, 127, 4888-4894), on the basis of photochemical and photophysical experiments. The new probe, AM-TG-betaGal, provides a dramatic fluorescence enhancement upon reaction with beta-galactosidase, and further hydrolysis of the ester moiety by ubiquitous intracellular esterases affords a hydrophilic product that is well retained within the cells without loss of fluorescence. We used a mouse tumor model to assess the practical utility of AM-TG-betaGal, after confirming that tumors in the model could be labeled with an avidin-beta-galactosidase conjugate. This conjugate was administered to the mice in vivo, followed by AM-TG-betaGal, and subsequent ex vivo fluorescence imaging clearly visualized intraperitoneal tumors as small as 200 microm. This strategy has potential clinical application, for example, in video-assisted laparoscopic tumor resection.     

In Situ Self‐Assembled Nanofibers Precisely Target Cancer‐Associated Fibroblasts for Improved Tumor Imaging

Tumor complexity makes the development of highly sensitive tumor imaging probes an arduous task. Here, we construct a peptide‐based near‐infrared probe that is responsive to fibroblast activation protein‐α (FAP‐α), and specifically forms nanofibers on the surface of cancer‐associated fibroblasts (CAFs) in situ. The assembly/aggregation‐induced retention (AIR) effect results in enhanced accumulation and retention of the probe around the tumor, resulting in a 5.5‐fold signal enhancement in the tumor 48 h after administration compared to that of a control molecule that does not aggregate. The probe provides a prolonged detectable window of 48 h for tumor diagnosis. The selective assembly of the probe results in a signal intensity over four‐ and fivefold higher in tumor than in the liver and kidney, respectively. With enhanced tumor imaging capability, this probe can visualize small tumors around 2 mm in diameter.     

~(64)Cu-NOTA-Herceptin的设计、活性测定及肿瘤靶向分子显像研究

利用双功能螯合剂2-[(4-异硫氰基苯基)甲基]-1,4,7-三氮杂环九烷-1,4,7-三乙酸(NCS-Bz-NOTA)对Herceptin单抗表面的氨基进行修饰获得了NOTA-Herceptin,通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)对该偶联物进行了表征.利用酶联免疫吸附测定了偶联前后Herceptin抗体效价的改变.利用新型正电子核素~(64)Cu标记,获得可用于肿瘤放射靶向精准诊疗的~(64)Cu-NOTA-Herceptin探针,其标记率为90%,放化纯度98%,比活度185 MBq/nmol.分别进行了该探针在HER2过表达胃癌细胞NCI-N87及HER2低表达胃癌细胞BGC823等肿瘤细胞中的摄取实验,测定了该探针的肿瘤特异性.建立了荷人胃癌BGC823裸鼠模型,通过微型正电子断层显像(Micro-PET)设备观察了探针在模型动物体内的代谢情况:在静脉注射7.4 MBq~(64)Cu-NOTA-Herceptin探针后,分别于4和60 h进行正电子断层显像(PET)的显像,观察到其在肿瘤部位的摄取有所富集,且随着代谢时间的延长,肝脏部位摄取得到明显降低.研究结果表明,~(64)Cu-NOTAHerceptin探针有望应用于肿瘤放射性靶向诊疗.     

“Turn-On” Activatable AIE Dots for Tumor Hypoxia Imaging

A hypoxia-responsive fluorescence probe of amphiphilic PEGylated azobenzene caged tetraphenylethene (TPE) for tumor cell imaging is reported; it possesses excellent solubility in aqueous medium due to the easy formation of micelles by self-assembly. The fluorescence resonance energy transfer (FRET) process ensures that the fluorescence of the azobenene caged AIE fluorogen is quenched efficiently. When cultured with tumor cells, the azo-bond is reduced under hypoxia conditions and the fluorescence of AIE fluorogen recovers dramatically. Besides using UV light, NIR light can also be used as the excited light resource to generate the fluorescence due to the two-photon fluorescence imaging process.     

肿瘤乏氧靶向响应的罗丹明荧光探针及其成像介导手术治疗

基于罗丹明的良好荧光性能, 经化学偶联反应制备并表征了一个偶氮乏氧特异响应的“Off-On”型荧光成像探针(FY-4). 从分子层面证实了其荧光“Off-On”性能和响应机制; 在L02正常细胞及4T1, HeLa和A549肿瘤细胞层面考察了其对受试细胞株的毒性和不同乏氧时间的荧光成像性能; 再利用4T1肿瘤模型, 分别以肿瘤原位注射和尾静脉注射的方式考察了其荧光成像性能, 并探究了其荧光成像介导切除肿瘤性能, 最后还考察了FY-4的生物安全性. 结果表明, FY-4有高的肿瘤乏氧靶向特异“关-开”响应的荧光成像差异显影及荧光成像介导切除肿瘤的潜能, 结合其良好的光物理性能、 生物安全性和明晰的给药时间等特性, 有望为生物医学荧光成像介导肿瘤切除提供新的研究工具.     

Near‐Infrared‐Emitting BODIPY‐trisDOTA111In as a Monomolecular Multifunctional Imaging Probe: From Synthesis to In Vivo Investigations

A new generation of monomolecular imaging probes (MOMIP) based on a distyryl‐BODIPY (BODIPY=boron‐dipyrromethene) coupled with three DOTA macrocycles has been prepared (DOTA=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid). The MOMIP presents good fluorescence properties and is very stable in serum. The bimodal probe was conjugated to trastuzumab, and an optical in vivo study showed high accumulation of the imaging agent at the tumor site. ¹¹¹In radiometallation of the bioconjugate was performed in high radiochemical yield, highlighting the potential of this new BODIPY‐chelators derivative as a bimodal imaging probe.     

An Unsymmetrical Squaraine-Based Activatable Probe for Imaging Lymphatic Metastasis by Responding to Tumor Hypoxia with MSOT and Aggregation-Enhanced Fluorescent Imaging

Optoacoustic imaging has great potential for preclinical research and clinical practice, and designing robust activatable optoacoustic probes for specific diseases is beneficial for its further development. Herein, an activatable probe has been developed for tumor hypoxia imaging. For this probe, indole and quinoline were linked on each side of an oxocyclobutenolate core to form an unsymmetrical squaraine. A triarylamine group was incorporated to endow the molecule with the aggregation enhanced emission (AEE) properties. In aqueous media, the squaraine chromophore aggregates into the nanoprobe, which specifically responds to nitroreductase and produces strong optoacoustic signals due to its high extinction coefficient, as well as prominent fluorescence emission as a result of its AEE feature. The nanoprobe was used to image tumor metastasis via the lymphatic system both optoacoustically and fluorescently. Moreover, both the fluorescence signals and three-dimensional multispectral optoacoustic tomography signals from the activated nanoprobe allow us to locate the tumor site and to map the metastatic route.     

An NIR Fluorescence Turn-on and MRl Bimodal Probe for Concurrent Real-time in vivo Sensing and Labeling of β-Galactosidase

To realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel β-galactosidase (β-gal) activated bimodal imaging probe that combines near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI) to enable real-time visualization of activity in living organisms. Upon β-gal activation, Gal-Cy-Gd-1 exhibits a remarkable 42-fold increase in NIR fluorescence intensity at 717 nm, allowing covalent labeling of adjacent target enzymes or proteins and avoiding molecular escape to promote probe accumulation at the tumor site. This fluorescence reaction enhances the longitudinal relaxivity by approximately 1.9 times, facilitating high-resolution MRI. The unique features of Gal-Cy-Gd-1 enable real-time and precise visualization of β-gal activity in live tumor cells and mice. The probe's utilization aids in identifying in situ ovarian tumors, offering valuable assistance in the precise removal of tumor tissue during surgical procedures in mice. The fusion of NIR fluorescence and MRI activation through self-immobilizing target enzymes or proteins provides a robust approach for visualizing β-gal activity. Moreover, this approach sets the groundwork for developing other activatable bimodal probes, allowing real-time in vivo imaging of enzyme activity and localization.     

Real‐Time Fluorescence Turn‐On Detection of Alkaline Phosphatase Activity with a Novel Perylene Probe

A tetracationic perylene probe (probe 2 ) was designed and synthesized. Probe 1 was used for the real‐time fluorescence turn‐on assay of alkaline phosphatase (ALP) activity and inhibitor screening. Probe 1 monomer fluorescence could be very efficiently quenched by ATP through the formation of an ATP/probe 1 complex. ALP triggered the degradation of ATP, the breakdown of the ATP/probe 1 complex, and the recovery of the probe 1 monomer fluorescence. In the presence of an ALP inhibitor, a decrease in fluorescence recovery was observed.     

A Dendron-Based Fluorescence Turn-On Probe for Tumor Detection

Specifically amplifying the emission signals of optical probes in tumors is an effective way to improve the tumor-imaging sensitivity and contrast. In this paper, the first case of dendron-based fluorescence turn-on probes mediated by a Förster resonance energy transfer (FRET) mechanism is reported. Dendrons up to the fourth generation with a hydrophilic oligo(ethylene glycol) scaffold are synthesized by a solid-phase synthesis strategy, and show precise and defect-free chemical structures. To construct the fluorescence turn-on probe, one Cy5.5 molecule is conjugated to the focal of a G3 dendron through a robust linkage and eight Black Hole Quencher 3 (BHQ-3) molecules are conjugated to its periphery through a PEG chain bearing a reductively cleavable disulfide linkage. By in vitro and in vivo experiments, it is demonstrated that the fluorescence of the dendron-based probe can be activated effectively and rapidly in the reductive environments of tumor cells and tissues, and the probe thus exhibits amplified tumor signals and weak normal tissue signals. Compared with the reported nanoscale turn-on probes, the dendron-based probe has several significant advantages, such as well-defined chemical structure, precisely controllable fluorophore/quencher conjugation sites and ratio, desirable chemical stability, and reproducible pharmacokinetic and pharmacological profiles, and is very promising in tumor detection.     

Excimer-based Activatable Fluorescent Sensor for Sensitive Detection of Alkaline Phosphatase

The accurate detection of related biomarkers at early stage is crucial in diagnosis and therapy of cancer. Small molecule fluorescence probes stand out acting as efficient tools to detect biomolecules, especially biomacromolecules. Herein, a new probe that features excimer formation has been designed to detect cancer-related biomarker alkaline phosphatase(ALP). The probe shows high sensitivity to ALP with a low limit detection of 0.13 U/L and high selectivity with resistance to the interference in the complex physiological system. Furthermore, this probe presents good biocompatibility with negligible cytotoxicity and displays remarkable cell imaging functions, which allows for its application in biosystems.Theoretical simulation validates the high affinity of the probe with ALP and depicts the details of the interaction modes, which provides the possible mechanisms underlying the efficient detection of the probe in atomic level. The study of synthesis, properties and applications of the activatable emissive excimers not only enriches the tools to detect ALP, but also provides a new strategy to comprehend the pathogenic mechanism of enzyme-related diseases.     

Polymer‐Templated Perylene‐Probe Noncovalent Self‐Assembly: A New Strategy for Label‐Free Ultrasensitive Fluorescence Turn‐On Biosensing

A new label‐free fluorescence turn‐on strategy for highly sensitive biosensing has been developed. A negatively charged perylene probe was synthesized. Polycations could induce aggregation of the perylene probe through noncovalent interactions and the fluorescence of the probe’s monomer was efficiently quenched. Upon addition of a single‐stranded nucleic acid, competitive binding of the negatively charged nucleic acid (a polyanion) to the cationic polymer resulted in the release of a monomer and thus a turn‐on fluorescence signal was detected. Without the use of any amplification techniques, a detection limit of 2 pM DNA was obtained. Based on these results, an assay strategy for the highly sensitive detection of alkaline phosphatase (ALP) activity has been demonstrated. λ Exonuclease (λ exo) could degrade 5′‐phosphorylated single‐stranded DNA. However, when the DNA sample was treated with ALP, the phosphate functional group was removed by ALP and it could no longer be degraded by λ exo. Binding of the DNA to the perylene probe–polycation complex resulted in a turn‐on fluorescence signal, which could be used for sensing of ALP. The method is highly sensitive, a limit of detection as low as 0.02 mU mL^?1 ALP was obtained. Our method is simple, convenient, highly sensitive, and inexpensive.     

Design strategy for a near-infrared fluorescence probe for matrix metalloproteinase utilizing highly cell permeable boron dipyrromethene

Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existing NIR fluorescence probes for matrix metalloproteinases (MMPs), which are tumor, atherosclerosis, and inflammation markers, have various disadvantages, especially as regards sensitivity. Here, we report a novel design strategy to obtain a NIR fluorescence probe that is rapidly internalized by free diffusion and well retained intracellularly after activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or nonpermeable NIR fluorescent dye as a F?rster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases.