Recent Advances in the Development of Non-Invasive Imaging Probes for Cancer Immunotherapy.

The last two decades have witnessed a major revolution in the field of tumor immunology including clinical progress using various immunotherapy strategies. These advances have highlighted the potential for approaches that harness the power of the immune system to fight against cancer. While cancer immunotherapies have shown significant clinical successes, patient responses vary widely due to the complex and heterogeneous nature of tumors and immune responses, calling for reliable biomarkers and therapeutic strategies to maximize the benefits of immunotherapy. Especially, stratifying responding individuals from non-responders during the early stages of treatment could help avoid long-term damage and tailor personalized treatments. In efforts to develop non-invasive means for accurately evaluating and predicting tumor response to immunotherapy, multiple affinity-based agents targeting immune cell markers and checkpoint molecules have been developed and advanced to clinical trials. In addition, researchers have recently turned their attention to substrate and activity-based imaging probes that can provide real-time, functional assessment of immune response to treatment. Here, we highlight some of those recently designed probes that image functional proteases as biomarkers of cancer immunotherapy with a focus on their chemical design and detection modalities and discuss challenges and opportunities for the development of imaging tools utilized in cancer immunotherapy.

Ratiometric Afterglow Luminescent Imaging of Matrix Metalloproteinase-2 Activity via an Energy Diversion Process.

Ratiometric afterglow luminescent (AGL) probes are attractive for in vivo imaging due to their high sensitivity and signal self-calibration function. However, there are currently few ratiometric AGL probes available for imaging enzymatic activity in living organisms. Here, we present an energy diversion (ED) strategy that enables the design of an enzyme-activated ratiometric AGL probe (RAG-RGD) for in vivo afterglow imaging. The ED process provides RAG-RGD with a radiative transition for an 'always on' 520-nm AGL signal (AGL520) and a cascade three-step energy transfer (ET) process for an 'off-on' 710-nm AGL signal (AGL710) in response to a specific enzyme. Using matrix metalloproteinase-2 (MMP-2) as an example, RAG-RGD shows a significant ~11-fold increase in AGL710/AGL520 toward MMP-2. This can sensitively detect U87MG brain tumors through ratiometric afterglow imaging of MMP-2 activity, with a high signal-to-background ratio and deep imaging depth. Furthermore, by utilizing the self-calibration effect of ratiometric imaging, RAG-RGD demonstrated a strong negative correlation between the AGL710/AGL520 value and the size of orthotopic U87MG tumor, enabling accurate monitoring of orthotopic glioma growth in vivo. This ED process may be applied for the design of other enzyme-activated ratiometric afterglow probes for sensitive afterglow imaging.

Activatable Nanoreporters for Real-Time Tracking of Macrophage Phenotypic States Associated with Disease Progression.

Diagnosis of inflammatory diseases is characterized by identifying symptoms, biomarkers, and imaging. However, conventional techniques lack the sensitivities and specificities to detect disease early. Here, it is demonstrated that the detection of macrophage phenotypes, from inflammatory M1 to alternatively activated M2 macrophages, corresponding to the disease state can be used to predict the prognosis of various diseases. Activatable nanoreporters that can longitudinally detect the presence of the enzyme Arginase 1, a hallmark of M2 macrophages, and nitric oxide, a hallmark of M1 macrophages are engineered, in real-time. Specifically, an M2 nanoreporter enables the early imaging of the progression of breast cancer as predicted by selectively detecting M2 macrophages in tumors. The M1 nanoreporter enables real-time imaging of the subcutaneous inflammatory response that rises from a local lipopolysccharide (LPS) administration. Finally, the M1-M2 dual nanoreporter is evaluated in a muscle injury model, where an initial inflammatory response is monitored by imaging M1 macrophages at the site of inflammation, followed by a resolution phase monitored by the imaging of infiltrated M2 macrophages involved in matrix regeneration and wound healing. It is anticipated that this set of macrophage nanoreporters may be utilized for early diagnosis and longitudinal monitoring of inflammatory responses in various disease models.

NIR-II Self-Luminous Molecular Probe for In Vivo Inflammation Tracking and Cancer PDT Effect Self-Evaluating.

Optical imaging in the second near-infrared (NIR-II, 900-1700 nm) window has been extensively investigated for bioimaging. However, a strong autofluorescence background from real-time excitation light significantly reduces the images' quality of NIR-II fluorescence (FL) imaging. To resolve this issue, a NIR-II self-luminous small molecule (CLPD) based on bioluminescence (BL) resonance energy transfer (BRET) mechanism is first developed. The reactive oxygen species (ROS) can trigger NIR-II BL and reduce the NIR-II FL signals of the CLPD simultaneously, enabling ROS-correlated ratiometric BL/FL imaging. CLPD is used for high-contrast NIR-II BL imaging of osteoarthritis as well as guiding the treatment process by ratiometric BL/FL imaging. Moreover, CLPD is applied for NIR-II BL imaging of tumor triggered by the generated ROS during PDT. A correlation between the ratiometric NIR-II BL/FL signal and tumor size is constructed, providing a trustworthy tool for early assessment of PDT effect. Overall, this study presents a novel NIR-II self-luminous small molecular probe for in vivo imaging and provides a strategy for design a self-evaluation system of therapeutic effect.

"Dual-Key-and-Lock" NIR-II NSCyanines Enable High-Contrast Activatable Phototheranostics in Extrahepatic Diseases.

Conventional cyanine dyes with a symmetric structure are "always-on", which can easily accumulate in the liver and display high liver background fluorescence, inevitably interfering the accurate diagnosis and therapy in extrahepatic diseases. We herein report a platform of NIR-II non-symmetric cyanine (NSCyanine) dyes by harnessing a non-symmetric strategy, which are extremely sensitive to pH/viscosity and can be activated via a "dual-key-and-lock" strategy. These NSCyanine dyes with a low pKa (<4.0) only show weak fluorescence at lysosome pH (key1), however, the fluorescence can be completely switched on and significantly enhanced by intracellular viscosity (key2) in disease tissues, exhibiting high target-to-liver ratios up to 19.5/1. Notably, high-contrast phototheranostics in extrahepatic diseases are achieved, including intestinal metastasis-imaging, acute gastritis-imaging, bacteria infected wound healing, and tumor ablation via targeted combined photothermal therapy and chemotherapy.

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

Fluorescence imaging in the second near-infrared (NIR-II) window holds great promise for in vivo visualization of amyloid-ß (Aß) 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 Aß plaques in AD-model mice. Among all the designed probes, DMP2 exhibits the highest affinity to Aß fibrils and can specifically activate its NIR-II fluorescence after binding to Aß 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 Aß plaques in in vivo AD-model mice. Thus, this study presents a potential agent for non-invasive imaging of Aß plaques and deep deciphering of AD progression.

Highly Bright Near-Infrared Chemiluminescent Probes for Cancer Imaging and Laparotomy.

Near-infrared (NIR) chemiluminescence imaging holds potential for sensitive imaging of cancer due to its low background; however, few NIR chemiluminophores are available, which share the drawback of low chemiluminescence quantum yields (ΦCL ). Herein, we report the synthesis of NIR chemiluminophores for cancer imaging and laparotomy. Molecular engineering of the electron-withdrawing group at the para-position of the phenol-dioxetane leads to a highly bright NIR chemiluminophore (DPT), showing the ΦCL (4.6×10-2  Einstein mol-1 ) that is 3 to 5-fold higher than existing NIR chemiluminophores. By caging the phenol group of DPT with a cathepsin B (CatB) responsive moiety, an activatable chemiluminescence probe (DPTCB ) is developed for real-time turn-on detection of deeply buried tumor tissues in living mice. Due to its high brightness, DPTCB permits accurate chemiluminescence-guided laparotomy.

A Renal-Clearable Activatable Molecular Probe for Fluoro-Photacoustic and Radioactive Imaging of Cancer Biomarkers.

In vivo simultaneous visualization of multiple biomarkers is critical to accurately diagnose disease and decipher fundamental processes at a certain pathological evolution, which however is rarely exploited. Herein, a multimodal activatable imaging probe (P-125 I) is reported with activatable fluoro-photoacoustic and radioactive signal for in vivo imaging of biomarkers (i.e., hepsin and prostate-specific membrane antigen (PSMA)) associated with prostate cancer diagnosis and prognosis. P-125 I contains a near-infrared (NIR) dye that is caged with a hepsin-cleavable peptide sequence and linked with a radiolabeled PSMA-targeted ligand (PSMAL). After systemic administration, P-125 I actively targets the tumor site via specific recognition between PSMA and PSMAL moiety and in-situ generates of activated fluoro-photoacoustic signal after reacting with hepsin to release the free dye (uncaged state). P-125 I achieves precisely early detection of prostate cancer and renal clearance to alleviate toxicity issues. In addition, the accumulated radioactive and activated photoacoustic signal of probe correlates well with the respective expression level of PSMA and hepsin, which provides valuable foreseeability for cancer progression and prognosis. Thus, this study presents a multimodal activatable probe for early detection and in-depth deciphering of prostate cancer.

Activatable Multimodal Probes for In Vivo Imaging and Theranostics.

Multimodal imaging, which harnesses two or more imaging modalities to produce complementary anatomical and molecular information of a living subject, has become as a powerful tool in both basic biomedical research and clinical diagnosis. The progresses in multimodal imaging are paralleled by the advances in multimodal probes, particularly activatable multimodal imaging probes that can generate concurrent switches in different imaging modality signals upon interaction with a molecular target. These probes are extremely promising for in vivo imaging. In this Minireview, we summarize the recent progress in activatable multimodal probes for in vivo imaging and cancer theranostics, focusing on their design principle, signal activation mechanism and biomedical applications. The current challenges and perspectives for future developments of activatable multimodal probes are also briefly discussed. We hope that this Minireview will provide inspiration for the design of other activatable multimodal probes for improving in vivo imaging and theranostics.

Spatiotemporally Controlled Formation and Rotation of Magnetic Nanochains In Vivo for Precise Mechanotherapy of Tumors.

Systemic cancer therapy is always accompanied with toxicity to normal tissue, which has prompted concerted efforts to develop precise treatment strategies. Herein, we firstly develop an approach that enables spatiotemporally controlled formation and rotation of magnetic nanochains in vivo, allowing for precise mechanotherapy of tumor. The nanochain comprised nanocomposites of pheophorbide-A (PP) modified iron oxide nanoparticle (IONP) and lanthanide-doped down-conversion NP (DCNP). In a permanent magnetic field, the nanocomposites would be aligned to form nanochain. Next, MnO2 NPs were subsequently administered to accumulate in tumor as suppliers of Mn2+ , which coordinates with PP to immobilize the nanochain. In a rotating magnetic field, the nanochain would rapidly rotate, leading to apoptosis/necrosis of tumor cell. The nanochain showed high T2 -MR and NIR-II fluorescence imaging signals, which facilitated guided therapy. The strategy has great potential in practical applications.

A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours.

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti-tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B-mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B-specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal-to-noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

Hemicyanine-Based Near-Infrared Activatable Probes for Imaging and Diagnosis of Diseases.

Molecular activatable probes with near-infrared (NIR) fluorescence play a critical role in in vivo imaging of biomarkers for drug screening and disease diagnosis. With structural diversity and high fluorescence quantum yields, hemicyanine dyes have emerged as a versatile scaffold for the construction of activatable optical probes. This Review presents a survey of hemicyanine-based NIR activatable probes (HNAPs) for in vivo imaging and early diagnosis of diseases. The molecular design principles of HNAPs towards activatable optical signaling against various biomarkers are discussed with a focus on their broad applications in the detection of diseases including inflammation, acute organ failure, skin diseases, intestinal diseases, and cancer. This progress not only proves the unique value of HNAPs in preclinical research but also highlights their high translational potential in clinical diagnosis.

Rational Design of a Highly Selective Near-Infrared Two-Photon Fluorogenic Probe for Imaging Orthotopic Hepatocellular Carcinoma Chemotherapy.

Selective fluorescence imaging of biomarkers in vivo and in situ for evaluating orthotopic hepatocellular carcinoma (HCC) chemotherapy remains a great challenge due to current imaging agents suffering from the potential interferences of other hydrolases. Herein, we observed that carbamate unit showed a high selectivity toward the HCC-related biomarker carboxylesterase (CE) for evaluation of treatment. A near-infrared two-photon fluorescent probe was developed to not only specially image CE activity in vivo and in situ but also target orthotopic liver tumor after systemic administration. The in vivo signals of the probe correlate well with tumor apoptosis, making it possible to evaluate the status of treatment. The probe enables the imaging of CE activity in situ with a high-resolution three-dimensional view for the first time. This study may promote advances in optical imaging approaches for precise imaging-guided diagnosis of HCC in situ and its evaluation of treatment.

Dual Ratiometric SERS and Photoacoustic Core-Satellite Nanoprobe for Quantitatively Visualizing Hydrogen Peroxide in Inflammation and Cancer.

Excessive production of oxidative species alters the normal redox balance and leads to diseases, such as chronic inflammation and cancer. Oxidative species are short-lived species, which makes direct, precise, and real-time measurements difficult. Herein, we report a novel core-satellite gold nanostructure for dual, ratiometric surface-enhanced Raman scattering (SERS) and photoacoustic (PA) imaging to enable the precise detection of inflammation/cancer-related H2 O2 . The combination of H2 O2 -activated second near-infrared (NIR-II) PA imaging and SERS imaging enables the differentiation between the inflamed region and normal tissue with high accuracy. The mesoporous silica shell of the nanoprobe could be used to deliver drugs to the target area to precisely treat disease. Therefore, this core-satellite nanostructure can not only quantitatively and precisely monitor H2 O2 produced in inflammation, tumor, and osteoarthritis in rabbits in real-time, but can also be used to track the progress of the anti-inflammatory treatment in real-time.

Molecular Chemiluminescent Probes with a Very Long Near-Infrared Emission Wavelength for in†Vivo Imaging.

Chemiluminescence imaging is imperative for diagnostics and imaging due to its intrinsically high sensitivity. To improve in vivo detection of biomarkers, chemiluminophores that simultaneously possess near-infrared (NIR) emission and modular structures amenable to construction of activatable probes are highly desired; however, these are rare. Herein, we report two chemiluminophores with record long NIR emission (>750 nm) via integration of dicyanomethylene-4H-benzothiopyran or dicyanomethylene-4H-benzoselenopyran with dioxetane unit. Caging of the chemiluminophores with different cleavable moieties produces NIR chemiluminescence probes (NCPs) that only produce signals upon reaction with reactive oxygen species or enzymes, for example, ß-galactosidase, with a tissue-penetration depth of up to 2 cm. Thus, this study provides NIR chemiluminescence molecular scaffolds applicable for in vivo turn-on imaging of versatile biomarkers in deep tissues.

Photoacoustic Imaging Probes Based on Tetrapyrroles and Related Compounds.

Photoacoustic imaging (PAI) is a rapidly evolving field in molecular imaging that enables imaging in the depths of ultrasound and with the sensitivity of optical modalities. PAI bases on the photoexcitation of a chromophore, which converts the absorbed light into thermal energy, causing an acoustic pressure wave that can be captured with ultrasound transducers, in generating an image. For in vivo imaging, chromophores strongly absorbing in the near-infrared range (NIR; > 680 nm) are required. As tetrapyrroles have a long history in biomedical applications, novel tetrapyrroles and inspired mimics have been pursued as potentially suitable contrast agents for PAI. The goal of this review is to summarize the current state of the art in PAI applications using tetrapyrroles and related macrocycles inspired by it, highlighting those compounds exhibiting strong NIR-absorption. Furthermore, we discuss the current developments of other absorbers for in vivo photoacoustic (PA) applications.

Smart Magnetic and Fluorogenic Photosensitizer Nanoassemblies Enable Redox-Driven Disassembly for Photodynamic Therapy.

Stimuli-responsive smart photosensitizer (PS) nanoassemblies that allow enhanced delivery and controlled release of PSs are promising for imaging-guided photodynamic therapy (PDT) of tumors. However, the lack of high-sensitivity and spatial-resolution signals and fast washout of released PSs from tumor tissues have impeded PDT efficacy in vivo. Herein, we report tumor targeting, redox-responsive magnetic and fluorogenic PS nanoassemblies (NP-RGD) synthesized via self-assembly of a cRGD- and disulfide-containing fluorogenic and paramagnetic small molecule (1-RGD) for fluorescence/magnetic resonance bimodal imaging-guided tumor PDT. NP-RGD show high r1 relaxivity but quenched fluorescence and PDT activity; disulfide reduction by glutathione (GSH) promotes efficient disassembly into a small-molecule probe (2-RGD) and an organic PS (PPa-SH), which could further bind with intracellular albumin, allowing prolonged retention and cascade activation of fluorescence and PDT to ablate tumors.

Activatable Molecular Probes for Second Near-Infrared Fluorescence, Chemiluminescence, and Photoacoustic Imaging.

Optical imaging plays a crucial role in biomedicine. However, due to strong light scattering and autofluorescence in biological tissue between 650-900 nm, conventional optical imaging often has a poor signal-to-background ratio and shallow penetration depth, which limits its ability in deep-tissue in vivo imaging. Second near-infrared fluorescence, chemiluminescence, and photoacoustic imaging modalities mitigate these issues by their respective advantages of minimized light scattering, eliminated external excitation, and ultrasound detection. To enable disease detection, activatable molecular probes (AMPs) with the ability to change their second near-infrared fluorescence, chemiluminescence, or photoacoustic signals in response to a biomarker have been developed. This Minireview summarizes the molecular design strategies, sensing mechanisms, and imaging applications of AMPs. The potential challenges and perspectives of AMPs in deep-tissue imaging are also discussed.

An Acid-Activatable Fluorescence Probe for Imaging Osteocytic Bone Resorption Activity in Deep Bone Cavities.

A rationally designed pH-activatable fluorescent probe (pHocas-RIS) has been used to measure localised pH levels in osteocytic lacunae in bone tissue. Conjugation of the moderate bone-binding drug risedronate to a pH-activatable BODIPY fluorophore enables the probe to penetrate osteocytic lacunae cavities that are embedded deep within the bone matrix. After injection of pHocas-RIS, any osteocytic lacunae caused by bone-resorbing osteocytes cause the probe to fluoresce in vivo, thus allowing imaging by intravital two-photon excitation microscopy. This pH responsive probe enabled the visualization of the bone mineralizing activities of acid producing osteocytes in real time, thus allowing the study of their central role in remodeling the bone-matrix in healthy and disease states.

Functional Imaging with Nucleic-Acid-Based Sensors: Technology, Application and Future Healthcare Prospects.

Timely monitoring and assessment of human health plays a crucial role in maintaining the wellbeing of our advancing society. In addition to medical tools and devices, suitable probe agents are crucial to assist such monitoring, either in passive or active ways (i.e., sensors) through inducible signals. In this review we highlight recent developments in activatable optical sensors based on nucleic acids. Sensing mechanisms and bio-applications of these nucleic acid sensors in ex vivo assays, intracellular or in vivo settings are described. In addition, we discuss the limitations of these sensors and how nanotechnology can complement/enhance sensor properties to promote translation into clinical applications.