Recent Advances in Activatable Probes for Molecular Imaging by Stimuli-Controlled Disassembly

Comprehensive Summary,Stimuli-controlled disassembly process has shown promise to direct delivery of probes and/or spatial-temporally control imaging signals for molecular imaging in vivo.Via the disassembly process,well defined nanoprobes with a stimulus-responsive moiety can be controllably converted into small-molecular imaging agents in response to a stimulus,leading to a switch in imaging signals.Moreover,the on-site released small-molecule probes could enhance penetration into the deep tissue for improved imaging of deep-seated molecular targets.Therefore,such a stimuli-controllable disassembly approach has been widely utilized to build activatable molecular imaging probes for the noninvasive detection of various molecular targets in living subjects.In this review article,we first briefly introduce the general principle of stimuli-controlled disassembly.We then summarize the activatable probes based on different internal or external stimulus that has been utilized to control disassembly process.Activatable probes by using multiple stimuli to control cascaded in situ self-assembly and disassembly processes are also discussed.Finally,we close with a conclusion of current challenges and perspective in this field.

Molecular imaging of enzyme activity in vivo using activatable probes

Precise measurement of enzyme activity in living systems with molecular imaging probes is becoming an important technique to unravel the functional roles of different enzymes in biological processes. Recent progress has been made in the development of a myriad of molecular imaging probes featuring different imaging modalities, including optical imaging, magnetic resonance imaging, nuclear imaging, and photoacoustic imaging, allowing for non-invasive detection of various enzyme activities in vivo with high sensitivity and high spatial resolution. Among these imaging probes, activatable or ＂smart＂ probes, whose imaging signal can be specifically switched from the ＂off＂ to ＂on＂ state upon interaction with a target enzyme, are particularly attractive due to their improved sensitivity and specificity. Here, recent advances in the development of activatable probes capable of imaging different enzyme activities in vivo are summarized based on different imaging modalities, and current challenges and future perspectives are discussed.

Alkaline Phosphatase-Initiated Sensitive Responsiveness of Activatable Probes to Hydrogen Sulfide for Accurate Cancer Imaging and Differentiation

Optical imaging with molecular probes is becoming an essential tool for advancing biological research and clinical applications.However,most currently available molecular probes show limited sensitivity,specificity,and accuracy due to their typical responsiveness to a single stimulation for biomarker-based imaging.In this study,we develop a novel molecular probe that shows alkaline phosphatase(ALP)-instructed sensitive responsiveness to hydrogen sulfide for accurate cancer imaging and differentiation.This designed probe in an aggregated state under physiological conditions bears negatively charged surfaces,giving poor optical response to H_(2)S.The ALP-mediated dephosphorylation reaction yields an assembled product with a positively charged surface,affording significantly aggregation-enhanced responsiveness to H_(2)S with light-up NIR fluorescence at 755 nm.Such charge reversal of assembled probe from negative to positive plays a vital role in allowing precise visualization and differentiation of cancers based on differences in ALP upregulation and H_(2)S content.We envisage that our charge-reversal strategy for multiple-parameter-activated molecule probes will facilitate boosting the specificity and precision of cancer imaging.

ACTIVATABLE SMART PROBES FOR MOLECULAR OPTICAL IMAGING AND THERAPY

Recent years have seen the design and implementation of many optical activatable smart probes.These probes are activatable because they change their optical properties and are smart because they can identify specific targets.This broad class of detection agents has allowed previously unperformed visualizations,facilitating the study of diverse biomolecules including enzymes,nucleic acids,ions and reactive oxygen species.Designed to be robust in an in vivo environment,these probes have been used in tissue culture cells and in live small animals.An emerging class of smart probes has been designed to harness the potency of singlet oxygen generating photosensitizers.Combining the discrimination of activatable agents with the toxicity of photosensitizers represents a new and powerful approach to disease treatment.This review highlights some applications of activatable smart probes with a focus on developments of the past decade.

A metabolic acidity-activatable calcium phosphate probe with fluorescence signal amplification capabilities for non-invasive imaging of tumor malignancy

Dysregulated energy metabolism has recently been recognized as an emerging hallmark of cancer.Tumor cells,which are characterized by abnormal glycolysis,exhibit a lower extracellular pH(6.5–7.0)than nor-mal tissues(7.2–7.4),providing a promising target for tumor-specific imaging and therapy.However,most pH-sensitive materials are unable to distinguish such a subtle pH difference owing to their wide and continuous pH-responsive range.In this study,we developed an efficient strategy for the fabrication of a tumor metabolic acidity-activatable calcium phosphate(CaP)fluorescent probe(termed MACaP9).Unlike traditional CaP-based biomedical nanomaterials,which only work within more acidic organelles,such as endosomes and lysosomes(pH 4.0–6.0),MACaP9 could not only specifically respond to the tumor extra-cellular pH but also rapidly convert pH variations into a distinct fluorescence signal to visually dis-tinguish tumor from normal tissues.The superior sensitivity and specificity of MACaP9 enabled high-contrast visualization of a broad range of tumors,as well as small tumor lesions.

Recent advances in the development of activatable multifunctional probes for in vivo imaging of caspase-3

Caspases are a family of proteases that play critical roles in controlling inflammation and cell death.Apoptosis is a caspase-3 mainly controlled behavior to avoid inflammation and damage to surrounding cells,whereas anomalistic cell apoptosis may be associated with many diseases.The detection and imaging of caspase-3 will be of great significance in evaluating the early therapeutic effect of tumors.Developing smart fluorescent probes may be helpful for the visualization of the rapeutic effect compared with "always on" probes.Thus,more and more works toward activatable fluorescent probes for caspase-3 imaging have been reported.In addition,multifunctional probes have also been designed to further improve the imaging of caspase-3.Herein,this review systematically summarized the representative wo rk of caspase-3 from the perspective of molecular design that it will play a guiding role in the design of probes that respond to caspase-3.Also,challenges and perspectives toward the field for imaging of cell apoptosis(caspase-3) are also discussed.

A pH-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration

We present here a pH-responsive activatable aptamer probe for targeted cancer imaging based on i-motif-driven conformation alteration. This pH-responsive activatable aptamer probe is composed of two single-stranded DNA. One was used for target recognition, containing a central, target specific aptamer sequence at the 3＇-end and an extension sequence at the 5＇-end with 5-carboxytetramethylrhodamine （TAMRA） label （denoted as strand A）. The other （strand ｜）, being competent to work on the formation of i-motif structure, contained four stretches of the cytosine （C） rich domain and was labeled with a Black Hole Quencher 2 （BHQ2） at the 3＇-end. At neutral or slightly alkaline pH, strand ｜ was hybridized to the extension sequence of strand A to form a double-stranded DNA probe, termed i-motif-based activatable aptamer probe （I-AAP）. Because of proximity- induced energy transfer, the I-AAP was in a ＂signal off＇ state. The slightly acidic pH enforced the strand I to form an intramo- lecular i-motif and then initiated the dehybridization of I-AAP, leading to fluorescence readout in the target recognition. As a demonstration, AS1411 aptamer was used for MCF-7 cells imaging. It was displayed that the I-AAP could be carried out for target cancer cells imaging after being activated in slightly acidic environment. The applicability of I-AAP for tumor tissues imaging has been also investigated by using the isolated MCF-7 tumor tissues. These results implied the I-AAP strategy is promising as a novel approach for cancer imaging.

An activatable ratiometric near-infrared fluorescent probe for hydrogen sulfide imaging in vivo

Ratiometric fluorescent probes hold great promise for in vivo imaging;however,stimuli-activatable ratiometric probes with fluorescence emissions in near-infrared(NIR)region are still very few.Herein,we report a hydrogen sulfide(H_2S)-activatable ratiometric NIR fluorescent probe(1-SPN)by integrating a H_2S-responsive NIR fluorescent probe 1 into a H_2S-inert poly[2,6-(4,4-bis-(2-ethylhexyl)-4 H-cyclopenta[2,1-b;3,4-b′]dithiophene)-alt-4,7(2,1,3-benzothiadiazole)](PCPDTBT)-based NIR semiconducting polymer nanoparticle(SPN).1-SPN shows"always on"PCPDTBT fluorescence at 830 nm and weak probe 1 fluorescence at 725 nm under excitation at 680 nm.The ratio of NIR fluorescence intensities between 725 and 830 nm(I_(725)/I_(830))is small.Upon interaction with H_2S,the fluorescence at 725 nm is rapidly switched on,resulting in a large enhancement of I_(725)/I_(830),which is allowed for sensitive visualization and quantification of H_2S concentrations in living cells.Taking advantage of enhanced tissue penetration depth of NIR fluorescence,1-SPN is also applied for real-time ratiometric fluorescence imaging of hepatic and tumor H_2S in living mice.This study demonstrates that activatable ratiometric NIR fluorescent probes hold great potential for in vivo imaging.

An activatable NIR-Ⅱ fluorescent probe for tracking heavy-metal ion and high-level salt-induced oxidative stress in plant sprouts

Humans and plants have become enfolded and inseparable.Abiotic stresses in particular oxidative stress caused by heavy-metal ions or high-level salt contamination deleteriously impact plants’growth process and have become a major threat to sustaining food security.Sprouting is the first step in plants’growth process.When plant sprouts endure oxidative stress induced by toxic heavy-metal ions or high-level salt,accelerated generation of reactive oxygen species(e.g.,H_(2)O_(2))occurs inside plant sprouts;hence in-situ H_(2)O_(2) in plant sprouts could serve as the in-vivo biomarker for tracking the oxidative stress in plant sprouts.Herein,we design an activatable probe CT-XA-H_(2)O_(2) to track the oxidative stress in plant sprouts via in vivo NIR-Ⅱ fluorescent imaging.In CT-XA-H_(2)O_(2),cyano-thiazole acts as the electron-accepting moiety and xanthane-aminodiphenyl as the electron-donating moiety,and dioxaborolane as the biomarker-responsive unit and fluorescence quencher.The probe CT-XA-H_(2)O_(2) shows weak fluorescent emission.When H_(2)O_(2) is present,the dioxaborolane in the probe is cleaved,consequently,the dye CT-XA-OH is generated and brings about significant fluorescent signals for detecting and imaging the in-situ biomarker.Moreover,the aminodiphenyl group endues the chromophore(the activated probe)with aggregation-induced emission characteristics,which ensures stronger fluorescence in the aggregated state in the aqueous milieu.The probe CT-XA-H_(2)O_(2) has been employed in the Cd^(2+)-ion or high-level salt(NaCl)induced oxidative stress models of soybean sprouts and peanut sprouts,and the experimental results evidently reveal the probe’s ability for in-situ biomarker-activatable in-vivo detection and imaging in the plants’sprouts.

“Dual-Lock-Dual-Key”Controlled Second Near-Infrared Molecular Probe for Specific Discrimination of Orthotopic Colon Cancer and Imaging-Guided Tumor Excision

Fluorescence imaging in the second infrared window(1000-1700 nm)has emerged as a promising approach to tumor diagnosis.However,the currently available second near-infrared(NIR-II)imaging agents are based on the“always on”modality or single biomarker activation,which are subject to limited imaging contrast,nonspecific response,and even false-positive diagnosis.Here,we developed a H2S/H+dual-stimuli responsive NIR-II fluorescent probe,WH-N3,for precise tumor delimitation and intraoperative fluorescence-guided surgical resection.WH-N3 itself is nonfluorescent,and it can only light up through synergistic activation by H2S and in the tumor acidic environment(TEM).Such a“duallock-dual-key”strategy-based activatable probe exhibited significantly higher tumor-to-normal tissue(T/N)ratios than the“always on”agent(ICG)and single parameter responsive counterpart probes in the imaging of colon tumors,which overexpresses H2S.WH-N3 was also able to visualize the tumor-derived endogenous H2S fluctuation and accurately differentiate tumor types based on H2S content discrepancy.More excitingly,under the guidance of the probe’s highly specific NIR-II fluorescence,a tiny orthotopic colon tumor with diameter down to 0.8 mm was facilely resected.We expect our dual-stimuli responsive strategy will contribute more reliable tools for specific discrimination and imaging-guided excision of tumor.

Gathering brings strength:How organic aggregates boost disease phototheranostics

Phototheranostics that concurrently and complementarily integrate real-time diagnosis and in situ therapeutic capabilities in one platform has become the advancing edge of precision medicine.Organic agents possess the merits of facile preparation,high purity,tunable photophysical property,good biocompatibility,and potential biodegradability,which have shown great promise for disease theranostics.This review summarizes the recent achievements of organic phototheranostic agents and applications,especially which rationally utilize energy dissipation pathways of Jablonski diagram to modulate the fluorescence emission,photoacoustic/photothermal production,and photodynamic processes.Of particular interest are the systems exhibiting huge differences in aggregate state as compared with the solution or single molecule form,during which the intramolecular motions play an important role in regulating the photophysical properties.The recent advances from such an aspect for biomedical applications including high-resolution imaging,activatable imaging and therapy,adaptive theranostics,image-guided surgery,immunotherapy,and afterglow imaging are discussed.A brief summary and perspective in this field are also presented.We hope this review will be helpful to the researchers interested in bioprobe design and theranostic applications,and inspire new insights into the linkage between aggregate science and biomedical field.