Enzyme-responsive, multi-lock optical probes for molecular imaging and disease theranostics.

Optical imaging is an indispensable tool for non-invasive visualization of biomolecules in living organisms, thereby offering a sensitive approach for disease diagnosis and image-guided disease treatment. Single-lock activatable optical probes (SOPs) that specifically switch on optical signals in the presence of biomarkers-of-interest have shown both higher detection sensitivity and imaging quality as compared to conventional "always-on" optical probes. However, such SOPs can still show "false-positive" results in disease diagnosis due to non-specific biomarker expression in healthy tissues. By contrast, multi-lock activatable optical probes (MOPs) that simultaneously detect multiple biomarkers-of-interest could improve detection specificity towards certain biomolecular events or pathological conditions. In this Review, we discuss the recent advancements of enzyme-responsive MOPs, with a focus on their biomedical applications. The higher detection specificity of MOPs could in turn enhance disease diagnosis accuracy and improve treatment efficacy in image-guided disease therapy with minimal toxicity in the surrounding healthy tissues. Finally, we discuss the current challenges and suggest future applications of MOPs.

Leveraging Long-Distance Singlet-Oxygen Transfer for Bienzyme-Locked Afterglow Imaging of Intratumoral Granule Enzymes.

Dual-locked activatable optical probes, leveraging the orthogonal effects of two biomarkers, hold great promise for the specific imaging of biological processes. However, their design approaches are limited to a short-distance energy or charge transfer mechanism, while the signal readout relies on fluorescence, which inevitably suffers from tissue autofluorescence. Herein, we report a long-distance singlet oxygen transfer approach to develop a bienzyme-locked activatable afterglow probe (BAAP) that emits long-lasting self-luminescence without real-time light excitation for the dynamic imaging of an intratumoral granule enzyme. Composed of an immuno-biomarker-activatable singlet oxygen (1O2) donor and a cancer-biomarker-activatable 1O2 acceptor, BAAP is initially nonafterglow. Only in the presence of both immune and cancer biomarkers can 1O2 be generated by the activated donor and subsequently diffuse toward the activated acceptor, resulting in bright near-infrared afterglow with a high signal-to-background ratio and specificity toward an intratumoral granule enzyme. Thus, BAAP allows for real-time tracking of tumor-infiltrating cytotoxic T lymphocytes, enabling the evaluation of cancer immunotherapy and the differentiation of tumor from local inflammation with superb sensitivity and specificity, which are unachievable by single-locked probes. Thus, this study not only presents the first dual-locked afterglow probe but also proposes a new design way toward dual-locked probes via reactive oxygen species transfer processes.

A Tandem-Locked Chemiluminescent Probe for Imaging of Tumor-Associated Macrophage Polarization.

Tumor-associated macrophages (TAMs) play a role in both pro- and anti-tumor functions; and the targeted polarization from M2 to M1 TAMs has become an effective therapy option. Although detection of M1 TAMs is imperative to assess cancer immunotherapeutic efficacy, existing optical probes suffer from shallow tissue penetration depth and poor specificity toward M1 TAMs. Herein, we report a tandem-locked NIR chemiluminescent (CL) probe for specific detection of M1 TAMs. Through a combined molecular engineering approach via both atomic alternation and introduction of electron-withdrawing groups, near-infrared (NIR) chemiluminophores are screened out to possess record-long emission (over 800 nm), record-high CL quantum yield (2.7 % einstein/mol), and prolonged half-life (7.7 h). Based on an ideal chemiluminophore, the tandem-locked probe (DPDGN) is developed to only activate CL signal in the presence of both tumour (γ-glutamyl transpeptidase) and M1 macrophage biomarkers (nitric oxide). Such a tandem-lock design ensures its high specificity towards M1 macrophages in the tumor microenvironment over those in normal tissues or peripheral blood. Thus, DPDGN permits noninvasive imaging and tracking of M1 TAM in the tumor of living mice during R837 treatment, showing a good correlation with ex vivo methods. This study not only reports a new molecular approach towards highly efficient chemiluminophores but also reveals the first tandem-locked CL probes for enhanced imaging specificity.

A Tandem-Locked Fluorescent NETosis Reporter for the Prognosis Assessment of Cancer Immunotherapy.

NETosis, the peculiar type of neutrophil death, plays important roles in pro-tumorigenic functions and inhibits cancer immunotherapy. Non-invasive real-time imaging is thus imperative for prognosis of cancer immunotherapy yet remains challenging. Herein, we report a Tandem-locked NETosis Reporter 1 (TNR1 ) that activates fluorescence signals only in the presence of both neutrophil elastase (NE) and cathepsin G (CTSG) for the specific imaging of NETosis. In the aspect of molecular design, the sequence of biomarker-specific tandem peptide blocks can largely affect the detection specificity towards NETosis. In live cell imaging, the tandem-locked design allows TNR1 to differentiate NETosis from neutrophil activation, while single-locked reporters fail to do so. The near-infrared signals from activated TNR1 in tumor from living mice were consistent with the intratumoral NETosis levels from histological results. Moreover, the near-infrared signals from activated TNR1 negatively correlated with tumor inhibition effect after immunotherapy, thereby providing prognosis for cancer immunotherapy. Thus, our study not only demonstrates the first sensitive optical reporter for noninvasive monitoring of NETosis levels and evaluation of cancer immunotherapeutic efficacy in tumor-bearing living mice, but also proposes a generic approach for tandem-locked probe design.

Artificial Urinary Biomarkers for Early Diagnosis of Acute Renal Allograft Rejection.

Acute renal allograft rejection (ARAR) after kidney transplantation associated with reduced graft survival and eventual graft failure is poorly diagnosed in hospitals. Here, we report the development of Artificial bioMarker Probes (AMPros) for sensitive urinalysis of ARAR in murine models. AMPros spontaneously go to the kidneys after systemic administration, specifically react with the prodromal immune biomarkers to activate their near-infrared fluorescence signals to report cell-mediated rejection, and efficiently undergo renal excretion into urine. Thus, AMPros enable convenient optical urinalysis that detects ARAR prior to histological manifestation of rejection, which is also earlier than current diagnostic methods measuring proinflammatory cytokines and peripheral blood lymphocyte mRNAs. Due to the high kidney specificity, AMPros-based urinalysis discriminates allograft rejection against other non-alloimmune specific diseases, which is unattainable by measurement of serological biomarkers. Such a noninvasive and sensitive urine test holds great promise in continuous monitoring of renal allograft conditions at low resource settings for timely clinical interventions.

A Semiconducting Iron-Chelating Nano-immunomodulator for Specific and Sensitized Sono-metallo-immunotherapy of Cancer.

Sono-immunotherapy holds great potential for deep tumor inhibition; however, smart sono-therapeutic agents to simultaneously eliminate 'domestic' tumor cells and regulate the 'community' tumor immune microenvironment have rarely been developed. Herein, we report a spatiotemporally controllable semiconducting iron-chelated nano-metallomodulator (SINM) for hypersensitive sono-metallo-immunotherapy of cancer. SINM consists of a semiconducting polymer (SP) backbone chelating iron ions (Fe3+ ) with thiophene-based Schiff base structure, and a hydrophilic side chain. Upon accumulation in tumors after systemic administration, SINM specifically arouses ferroptosis and M1 macrophage polarization due to its response toward the tumor redox environment; meanwhile, the chelation of Fe3+ enhances the sono-sensitizing effect of SPs, leading to enhanced generation of reactive oxygen species for immunogenic cell death. Such combined sonodynamic metallo-immunotherapy of SINM efficiently ablates deep tumor and spatiotemporally regulates immunophenotypes.

Smart Nano-PROTACs Reprogram Tumor Microenvironment for Activatable Photo-metabolic Cancer Immunotherapy.

Protease inhibitors can modulate intratumoral metabolic processes to reprogram the immunosuppressive tumor microenvironment (TME), which however suffer from the limited efficacy and off-targeted side effects. We report smart nano-proteolysis targeting chimeras (nano-PROTACs) with phototherapeutic ablation and cancer-specific protein degradation to reprogram the TME for photo-metabolic cancer immunotherapy. This nano-PROTAC has a semiconducting polymer backbone linked with a cyclooxygenase 1/2 (COX-1/2)-targeting PROTAC peptide (CPP) via a cathepsin B (CatB)-cleavable segment. CPP can be activated by the tumor-overexpressed CatB to induce the degradation of COX-1/2 via the ubiquitin-proteasome system. The persistent degradation of COX-1/2 depletes their metabolite prostaglandin E2 which is responsible for activation of immune suppressor cells. Such a smart PROTAC strategy synergized with phototherapy specifically reprograms the immunosuppressive TME and reinvigorates antitumor immunity.

Chemiluminescent Probes with Long-Lasting High Brightness for In Vivo Imaging of Neutrophils.

Real-time optical imaging of immune cells can contribute to understanding their pathophysiological roles, which still remains challenging. Current sensitive chemiluminophores have issues of short half-lives and low brightness, limiting their ability for in vivo longitudinal monitoring of immunological processes. To tackle these issues, we report benzoazole-phenoxyl-dioxetane (BAPD)-based chemiluminophores with intramolecular hydrogen bonding for in vivo imaging of neutrophils. Compared with the classical counterpart, chemiluminescence half-lives and brightness of BAPDs in the aqueous solution are increased by ∼ 33- and 8.2-fold, respectively. Based on the BAPD scaffold, a neutrophil elastase-responsive chemiluminescent probe is developed for real-time imaging of neutrophils in peritonitis and psoriasis mouse models. Our study provides an intramolecular hydrogen bonding molecular design for improving the performance of chemiluminophores in advanced imaging applications.

Renal-Clearable Molecular Probe for Near-Infrared Fluorescence Imaging and Urinalysis of SARS-CoV-2.

Despite the importance of rapid and accurate detection of SARS-CoV-2 in controlling the COVID-19 pandemic, current diagnostic methods are static and unable to distinguish between viable/nonviable virus or directly reflect viral replication activity. Real-time imaging of protease activity specific to SARS-CoV-2 can overcome these issues but remains lacking. Herein, we report a near-infrared fluorescence (NIRF) activatable molecular probe (SARS-CyCD) for detection of SARS-CoV-2 protease in living mice. The probe comprises a hemicyanine fluorophore caged with a protease peptide substrate and a cyclodextrin unit, which function as an NIRF signaling moiety and a renal-clearable enabler, respectively. The peptide substrate of SARS-CyCD can be specifically cleaved by SARS-CoV-2 main protease (Mpro), resulting in NIRF signal activation and liberation of the renal-clearable fluorescent fragment (CyCD). Such a design not only allows sensitive detection of Mpro in the lungs of living mice after intratracheal administration but also permits optical urinalysis of SARS-CoV-2 infection. Thus, this study presents an in vivo sensor that holds potential in preclinical high-throughput drug screening and clinical diagnostics for respiratory viral infections.

Charge-Reversal Polymer Nano-modulators for Photodynamic Immunotherapy of Cancer.

Nanomedicine can regulate the balance between cytotoxic T lymphocytes (CTLs) and suppressive regulatory T lymphocytes (Tregs), which however has been rarely exploited for cancer immunotherapy. We report a charge-reversal polymer nano-modulator (SPDMC N) activated by tumor microenvironment (TME) for photodynamic immunotherapy of cancer. SPDMC N is constructed by conjugating an immunomodulator (demethylcantharidin, DMC) to the side chains of a photodynamic polymer via an acid-liable linker. The negative charge of SPDMC N ensures its high stability in blood circulation and ideal tumor accumulation; exposure to acidic TME reverses its surface charge to positive, enhancing tumor penetration and locally releasing DMC. Upon near-infrared photoirradiation, SPDMC N generates singlet oxygen to ablate tumors and promote maturation of dendritic cells. Released DMC inhibits protein phosphatase 2 (PP2A) activity and decreases Tregs differentiation. Such combinational action induces a sharp increase in CTL/Treg ratio in TME and effectively inhibits both primary and distant tumors in living mice.

Multiplex Optical Urinalysis for Early Detection of Drug-Induced Kidney Injury.

Drug-induced kidney injury (DIKI) is a significant contributor of both acute and chronic kidney injury and remains a major concern in drug development and clinical care. However, current clinical diagnostic methods often fail to accurately and timely detect nephrotoxicity. This study reports the development of activatable molecular urinary reporters (MURs) that are able to specifically detect urinary biomarkers including γ-glutamyl transferase (GGT), alanine aminopeptidase (AAP), and N-acetyl-ß-d-glucosaminidase (NAG). By virtue of their discrete absorption and emission properties, the mixture of MURs can serve as a cocktail sensor for multiplex optical urinalysis in the mouse models of drug-induced acute kidney injury (AKI) and chronic kidney disease (CKD). The MURs cocktail not only detects nephrotoxicity earlier than the tested clinical diagnostic methods in drug-induced AKI and CKD mice models, but also possesses a higher diagnostic accuracy. Therefore, MURs hold great promise for detection of kidney function in both preclinical drug screening and clinical settings.

An Activatable Polymeric Reporter for Near-Infrared Fluorescent and Photoacoustic Imaging of Invasive Cancer.

Discriminative detection of invasive and noninvasive breast cancers is crucial for their effective treatment and prognosis. However, activatable probes able to do so in vivo are rare. Herein, we report an activatable polymeric reporter (P-Dex) that specifically turns on near-infrared (NIR) fluorescent and photoacoustic (PA) signals in response to the urokinase-type plasminogen activator (uPA) overexpressed in invasive breast cancer. P-Dex has a renal-clearable dextran backbone that is linked with a NIR dye caged with an uPA-cleavable peptide substrate. Such a molecular design allows P-Dex to passively target tumors, activate NIR fluorescence and PA signals to effectively distinguish invasive MDA-MB-231 breast cancer from noninvasive MCF-7 breast cancer, and ultimately undergo renal clearance to minimize the toxicity potential. Thus, this polymeric reporter holds great promise for the early detection of malignant breast cancer.

Unimolecular Chemo-fluoro-luminescent Reporter for Crosstalk-Free Duplex Imaging of Hepatotoxicity.

Real-time multiplex imaging is imperative to biology and diagnosis but remains challenging for optical modality. Herein, a unimolecular chemo-fluoro-luminescent reporter (CFR) is synthesized for duplex imaging of drug-induced hepatotoxicity (DIH), a long-term medical concern. CFR simultaneously detects superoxide anion (O2•-) and caspase-3 (casp3) through respective activation of its independent chemiluminescence and near-infrared fluorescence channels. Such a crosstalk-free duplex imaging capability of CFR enables longitudinal measurement of two correlated biomolecular events (oxidative stress and cellular apoptosis) during the progression of DIH, identifying O2•- as an earlier biomarker for detection of DIH both in vitro and in vivo. Moreover, CFR detects DIH 17.5 h earlier than histological changes. Thus, our study not only develops a sensitive optical reporter for early detection of DIH but also provides a general molecular design strategy for duplex imaging.

Recent Advances in Cell Membrane-Camouflaged Nanoparticles for Cancer Phototherapy.

Phototherapy including photothermal therapy (PTT) and photodynamic therapy (PDT) employs phototherapeutic agents to generate heat or cytotoxic reactive oxygen species (ROS), and has therefore garnered particular interest for cancer therapy. However, the main challenges faced by conventional phototherapeutic agents include easy recognition by the immune system, rapid clearance from blood circulation, and low accumulation in target sites. Cell-membrane coating has emerged as a potential way to overcome these limitations, owing to the abundant proteins on the surface of cell membranes that can be inherited to the cell membrane-camouflaged nanoparticles. This review summarizes the recent advances in the development of biomimetic cell membrane-camouflaged nanoparticles for cancer phototherapy. Different sources of cell membranes can be used to coat nanoparticles uisng different coating approaches. After cell-membrane coating, the photophysical properties of the original phototherapeutic nanoparticles remain nearly unchanged; however, the coated nanoparticles are equipped with additional physiological features including immune escape, in vivo prolonged circulation time, or homologous targeting, depending on the cell sources. Moreover, the coated cell membrane can be ablated from phototherapeutic nanoparticles under laser irradiation, leading to drug release and thus synergetic therapy. By combining other supplementary agents to normalize tumor microenvironment, cell-membrane coating can further enhance the therapeutic efficacy against cancer.

Recent Advances of Molecular Optical Probes in Imaging of ß-Galactosidase.

ß-Galactosidase (ß-Gal), as a lysosomal hydrolytic enzyme, plays an important physiological role in catalyzing the hydrolysis of glycosidic bonds which convert lactose into galactose. Moreover, upregulation of ß-Gal is often correlated with the occurrence of primary ovarian cancers and cell senescence. Thereby, detection of ß-Gal activity is relevant to cancer diagnosis. Optical imaging possesses high spatial and temporal resolution, high sensitivity, and real-time imaging capability. These properties are beneficial for the detection of ß-Gal in living systems. This Review summarizes the recent progress in development of molecular optical probes for near-infrared fluorescence (NIRF), bioluminescence (BL), chemiluminescence (CL), or photoacoustic (PA) imaging of ß-Gal in biological systems. The challenges and opportunities in the probe design for detection of ß-Gal are also discussed.

A Renal-Clearable Duplex Optical Reporter for Real-Time Imaging of Contrast-Induced Acute Kidney Injury.

Despite its high morbidity and mortality, contrast-induced acute kidney injury (CIAKI) remains a diagnostic dilemma because it relies on in vitro detection of insensitive late-stage blood and urinary biomarkers. We report the synthesis of an activatable duplex reporter (ADR) for real-time in vivo imaging of CIAKI. ADR is equipped with chemiluminescence and near-infrared fluorescence (NIRF) signaling channels that can be activated by oxidative stress (superoxide anion, O2.- ) and lysosomal damage (N-acetyl-ß-d-glucosaminidase, NAG), respectively. By virtue of its high renal clearance efficiency (80 % injected doses after 24 h injection), ADR detects sequential upregulation of O2.- and NAG in the kidneys of living mice prior to a significant decrease in glomerular filtration rate (GFR) and tissue damage in the course of CIAKI. ADR outperforms the typical clinical assays and detects CIAKI at least 8 h (NIRF) and up to 16 h (chemiluminescence) earlier.

Synthesis of PEGylated Semiconducting Polymer Amphiphiles for Molecular Photoacoustic Imaging and Guided Therapy.

Semiconducting polymer nanoparticles (SPNs) have been used as a new class of photonic materials with great potential in biomedical applications, but their synthetic method is limited to nanoprecipitation. Semiconducting polymer amphiphiles (SPAs) that can spontaneously self-assemble into nanoparticles are ideal alternatives for SPNs. Depending on their backbone structures, SPAs with different optical properties can be developed into nanoprobes for molecular imaging applications such as photoacoustic (PA) and fluorescence imaging as well as photothermal therapy. In this Concept, recent studies on the synthesis of SPAs for PA imaging and guided cancer therapy are summarized. The effect of grafting density on the optical properties of SPAs is discussed, and the nanoparticle sizes of SPAs can be reduced by utilization of a short semiconducting oligomer. Moreover, SPAs can be developed into PA theranostic platform and activatable PA nanoprobes. These studies demonstrate that SPAs are promising for advanced molecular imaging and therapy applications.

Near-Infrared Chemiluminescence Imaging of Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) has a high prevalence but is poorly managed for cancer patients due to the lack of reliable and sensitive diagnostic techniques. Molecular optical imaging can provide a noninvasive way for real-time monitoring of CIPN; However, this is not reported, likely due to the absence of optical probes capable of imaging deep into the spinal canal and possessing sufficient sensitivity for minimal dosage through local injection into the dorsal root ganglia. Herein, a near-infrared (NIR) chemiluminophore (MPBD) with a chemiluminescence quantum yield higher than other reported probes is synthesized and a NIR activatable chemiluminescent probe (CalCL) is developed for in vivo imaging of CIPN. CalCL is constructed by caging MPBD with calpain-cleavable peptide moiety while conjugating polyethylene glycol chain to endow water solubility. Due to the deep-tissue penetration of chemiluminescence and specific turn-on response of CalCL toward calpain (a hallmark of CIPN), it allows for sensitive detection of paclitaxel-mediated CIPN in living mice, which is unattainable by fluorescence imaging. This study thus not only develops a highly efficient chemiluminescent probe, but also presents the first optical imaging approach toward high-throughput screening of neurotoxic drugs.

Ingestible Artificial Urinary Biomarker Probes for Urine Test of Gastrointestinal Cancer.

Although colorectal cancer diagnosed at an early stage shows high curability, methods simultaneously possessing point-of-care testing ability and high sensitivity are limited. Here, an orally deliverable biomarker-activatable probe (termed as HATS) for early detection of orthotopic tumors via remote urinalysis is presented. To enable its oral delivery to the colon, HATS is designed to have remarkable resistance to acidity and digestive enzymes in the stomach and small intestine and negligible intestinal absorption. Upon reaction with a cancer biomarker in the colon segment, HATS releases a small fragment of tetrazine that can transverse the intestinal barrier, enter blood circulation, and ultimately undergo renal clearance to urine. Subsequently, the urinary tetrazine fragment is detected by bioorthogonal reaction with trans-cyclooctene-caged resorufin (TCO-Reso) to afford a rapid and specific fluorescence enhancement of TCO-Reso. Such signal readout is correlated with the urinary tetrazine concentration and thus measures the level of cancer biomarkers in the colon. HATS-based optical urinalysis detects orthotopic colon tumors two weeks earlier than clinical serological tests and can be developed to a point-of-care paper test. Thereby, HATS-based urinalysis provides a non-invasive and sensitive approach to cancer screening at low-resource settings.

Activatable near-infrared probes for the detection of specific populations of tumour-infiltrating leukocytes in vivo and in urine.

Tracking the immune microenvironment of tumours is essential for understanding the mechanisms behind the effectiveness of cancer immunotherapies. Molecular imaging of tumour-infiltrating leukocytes (TILs) can be used to non-invasively monitor the tumour immune microenvironment, but current imaging agents do not distinguish TILs from leukocytes resident in other tissues. Here we report a library of activatable molecular probes for the imaging, via near-infrared fluorescence, of specific TILs (including M1 macrophages, cytotoxic T lymphocytes and neutrophils) in vivo in real time and also via excreted urine, owing to the probes' renal clearance. The fluorescence of the probes is activated only in the presence of both tumour and leukocyte biomarkers, which allows for the imaging of populations of specific TILs in mouse models of cancers with sensitivities and specificities similar to those achieved via flow-cytometric analyses of biopsied tumour tissues. We also show that the probes enable the non-invasive evaluation of the immunogenicity of different tumours, the dynamic monitoring of responses to immunotherapies and the accurate prediction of tumour growth under various treatments.