Despite the recent advances in understanding the mechanisms of olfaction, no tools are currently available to noninvasively identify loss of smell. Because of the substantial increase in patients presenting with coronavirus disease 2019-related loss of smell, the pandemic has highlighted the urgent need to develop quantitative methods. Methods: Our group investigated the use of a novel fluorescent probe named Tsp1a-IR800P as a tool to diagnose loss of smell. Tsp1a-IR800P targets sodium channel 1.7, which plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. Results: Intuitively, we have identified that conditions leading to loss of smell, including chronic inflammation and coronavirus disease 2019, correlate with the downregulation of sodium channel 1.7 expression in the olfactory epithelium, both at the transcript and at the protein levels. We demonstrated that lower Tsp1a-IR800P fluorescence emissions significantly correlate with loss of smell in live animals-thus representing a potential tool for its semiquantitative assessment. Currently available methods rely on delayed subjective behavioral studies. Conclusion: This method could aid in significantly improving preclinical and clinical studies by providing a way to objectively diagnose loss of smell and therefore aid the development of therapeutic interventions.

BACKGROUND: In the treatment of oral cavity cancer, margin status is one of the most critical prognostic factors. Positive margins are associated with higher local recurrence and lower survival rates. Therefore, the universal goal of oral surgical oncology is to achieve microscopically clear margins. Near-infrared fluorescence guided surgery (FGS) could improve surgical resection using fluorescent probes. αVβ6 integrin has shown great potential for cancer targeting due to its overexpression in oral cancers. Red fluorescent contrast agent IRDye 680 coupled with anti-αVβ6 peptide (IRDye-A20) represents an asset to improve FGS of oral cancer. This study investigates the potential of IRDye-A20 as a selective imaging agent in 3D three-dimensional tongue cancer cells.
METHODS: αVβ6 integrin expression was evaluated by RT-qPCR and Western Blotting in 2D HSC-3 human tongue cancer cells and MRC-5 human fibroblasts. Targeting ability of IRDye-A20 was studied in both cell lines by flow cytometry technique. 3D tumor spheroid models, homotypic (HSC-3) and stroma-enriched heterotypic (HSC-3/MRC-5) spheroids were produced by liquid overlay procedure and further characterized using (immuno)histological and fluorescence-based techniques. IRDye-A20 selectivity was evaluated in each type of spheroids and each cell population.
RESULTS: αVβ6 integrin was overexpressed in 2D HSC-3 cancer cells but not in MRC-5 fibroblasts and consistently, only HSC-3 were labelled with IRDye-A20. Round shaped spheroids with an average diameter of 400 μm were produced with a final ratio of 55%/45% between HSC-3 and MRC-5 cells, respectively. Immunofluorescence experiments demonstrated an uniform expression of αVβ6 integrin in homotypic spheroid, while its expression was restricted to cancer cells only in heterotypic spheroid. In stroma-enriched 3D model, Cytokeratin 19 and E-cadherin were expressed only by cancer cells while vimentin and fibronectin were expressed by fibroblasts. Using flow cytometry, we demonstrated that IRDye-A20 labeled the whole homotypic spheroid, while in the heterotypic model all cancer cells were highly fluorescent, with a negligible fluorescence in fibroblasts.
CONCLUSIONS: The present study demonstrated an efficient selective targeting of A20FMDV2-conjugated IRDye 680 in 3D tongue cancer cells stroma-enriched spheroids. Thus, IRDye-A20 could be a promising candidate for the future development of the fluorescence-guided surgery of oral cancers.

Fibroblast activation protein-α (FAP) plays a crucial role in various physiological and pathological processes, making it a key target for cancer diagnostics and therapeutics. However, in vivo detection of FAP activity with fluorogenic probes remains challenging due to the rapid diffusion and clearance of fluorescent products from the target. Herein, we developed a self-immobilizing near-infrared (NIR) fluorogenic probe, Hcy-CF2H-PG, by introducing a difluoromethyl group to FAP substrate-caged NIR fluorophore. Upon selective activation by FAP, the fluorescence of Hcy-CF2H-PG was triggered, followed by the covalent labelling of FAP. Hcy-CF2H-PG demonstrated significantly improved sensitivity, selectivity, and long-lasting labelling capacity for FAP both in vitro and in vivo, compared to that of non-immobilized probes. This represents a noteworthy advancement in FAP detection and cancer diagnostics within complex physiological systems.

Neurodegenerative disorders, including Alzheimer\'s disease (AD) and Parkinson\'s disease (PD), represent debilitating conditions with complex, poorly understood pathologies. Epichaperomes, pathologic protein assemblies nucleated on key chaperones, have emerged as critical players in the molecular dysfunction underlying these disorders. In this study, we introduce the synthesis and characterization of clickable epichaperome probes, PU-TCO, positive control, and PU-NTCO, negative control. Through comprehensive in vitro assays and cell-based investigations, we establish the specificity of the PU-TCO probe for epichaperomes. Furthermore, we demonstrate the efficacy of PU-TCO in detecting epichaperomes in brain tissue with a cellular resolution, underscoring its potential as a valuable tool for dissecting single-cell responses in neurodegenerative diseases. This clickable probe is therefore poised to address a critical need in the field, offering unprecedented precision and versatility in studying epichaperomes and opening avenues for novel insights into their role in disease pathology.

Enzyme-activatable drug delivery systems have been developed for cancer diagnosis and therapy. However, targeted intracellular drug delivery is a challenge for precisely tumor imaging and therapy due to the increased stability of copolymer nanoparticles (NPs) is accompanied by a notable decrease in enzyme degradation. Herein, disulfide bond was designed as an enzyme-activatable molecular switch of SS-P(G2)2/DOX NPs. The copolymer NPs consists of polyvinylpyrrolidone (PVP) with disulfide bonds in the center and enzyme-degradable peptide dendrites (Phe-Lys) to form dendritic-linear-dendritic triblock copolymers (TBCs). The amphiphilic TBCs could be split into two identical amphiphilic diblock copolymers (DBCs) by glutathione (GSH) in cancer cells specifically while maintaining the same hydrophilic-lipophilic equilibrium. This structural transformation significantly reduced the stability of copolymer NPs and enhanced sensitivity of DOX release by cathepsin B-activated. Subsequently, the released DOX acted as an indicator of fluorescence imaging and chemotherapy drug for cancer cells. The polymeric NPs achieved excellent drug-loaded stability and prolonged blood circulation in vivo, and realized fluorescence imaging and specific cancer cell killing capabilities by responding to the overexpression of GSH and cathepsin B in tumor cells. Furthermore, the copolymer NPs demonstrated excellent blood compatibility and biosafety. Therefore, a novel strategy based on one tumor marker acting as the switch for another tumor microenvironment responsive drug delivery system could be designed for tumor intracellular imaging and chemotherapy.

Photothermal therapy (PTT), which uses the high thermal conversion ability of photothermal agents to ablate tumor cells at high temperatures, has gained significant attention because it has the advantages of high selectivity and specificity, precise targeting of tumor sites, and low invasiveness and trauma. However, PTT guided by the NIR-I has limitations in tissue penetration depth, resulting in limited imaging monitoring and therapeutic effects on deep-seated tumor tissues. Moreover, nanoparticles are easily cleared by the immune system and difficult to passively target tumor sites during the process of treatment. To address these issues, we prepared nanoparticles using NIR-II dyes IR1048 and DSPE-PEG-OH and further encapsulated them in red blood cell membranes derived from mice. These biomimetic nanoparticles, called RDIR1048, showed reduced clearance by the immune system and had long circulation characteristics. They effectively accumulated at tumor sites, and strong fluorescence could still be observed at the tumor site 96 h after administration. Furthermore, through mouse thermal imaging experiments, we found that RDIR1048 exhibited good PTT ability. When used in combination with an immune checkpoint inhibitor, anti-PD-L1 antibodies, it enhanced the immunogenic cell death of tumor cells caused by PTT and improved the therapeutic effect of immunotherapy, which demonstrated good therapeutic efficacy in the treatment of tumor-bearing mice. This study provides a feasible basis for the future development of NIR-II nanoparticles with long circulation properties.

The shape of a cell as defined by its membrane can be closely associated with its physiological state. For example, the irregular shapes of cancerous cells and elongated shapes of neuron cells often reflect specific functions, such as cell motility and cell communication. However, it remains unclear whether and which cell shape descriptors can characterize different cellular physiological states. In this study, 12 geometric shape descriptors for a three-dimensional (3D) object were collected from the previous literature and tested with a public dataset of ~400,000 independent 3D cell regions segmented based on fluorescent labeling of the cell membranes in Caenorhabditis elegans embryos. It is revealed that those shape descriptors can faithfully characterize cellular physiological states, including (1) cell division (cytokinesis), along with an abrupt increase in the elongation ratio; (2) a negative correlation of cell migration speed with cell sphericity; (3) cell lineage specification with symmetrically patterned cell shape changes; and (4) cell fate specification with differential gene expression and differential cell shapes. The descriptors established may be used to identify and predict the diverse physiological states in numerous cells, which could be used for not only studying developmental morphogenesis but also diagnosing human disease (e.g., the rapid detection of abnormal cells).

Throughout the central nervous system, the spinal cord plays a very important role, namely, transmitting sensory and motor information inwardly so that it can be processed by the brain. There are many different ways this structure can be damaged, such as through traumatic injury or surgery, such as scoliosis correction, for instance. Consequently, damage may be caused to the nervous system as a result of this. There is no doubt that optical devices such as microscopes and cameras can have a significant impact on research, diagnosis, and treatment planning for patients with spinal cord injuries (SCIs). Additionally, these technologies contribute a great deal to our understanding of these injuries, and they are also essential in enhancing the quality of life of individuals with spinal cord injuries. Through increasingly powerful, accurate, and minimally invasive technologies that have been developed over the last decade or so, several new optical devices have been introduced that are capable of improving the accuracy of SCI diagnosis and treatment and promoting a better quality of life after surgery. We aim in this paper to present a timely overview of the various research fields that have been conducted on optical devices that can be used to diagnose spinal cord injuries as well as to manage the associated health complications that affected individuals may experience.

Second near-infrared (NIR-II) fluorescence imaging is the most advanced imaging fidelity method with extraordinary penetration depth, signal-to-background ratio, biocompatibility, and targeting ability. It is currently booming in the medical realm to diagnose tumors and is being widely applied for fluorescence-imaging-guided tumor surgery. To efficiently execute this modern imaging modality, scientists have designed various probes capable of showing fluorescence in the NIR-II window. Here, we update the state-of-the-art NIR-II fluorescent probes in the most recent literature, including indocyanine green, NIR-II emissive cyanine dyes, BODIPY probes, aggregation-induced emission fluorophores, conjugated polymers, donor-acceptor-donor dyes, carbon nanotubes, and quantum dots for imaging-guided tumor surgery. Furthermore, we point out that the new materials with fluorescence in NIR-III and higher wavelength range to further optimize the imaging results in the medical realm are a new challenge for the scientific world. In general, we hope this review will serve as a handbook for researchers and students who have an interest in developing and applying fluorescent probes for NIR-II fluorescence-imaging-guided surgery and that it will expedite the clinical translation of the probes from bench to bedside.

A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living cells. However, existing FA-HCR methods usually face some problems, such as a complicated design and significant background leakage, which greatly limit their application. Herein, we developed an FA-centered HCR (FAC-HCR) method based on a remote toehold-mediated strand displacement reaction. Compared to traditional HCRs mediated by four hairpin probes (HPs) and two HPs, the FAC-HCR displayed significantly decreased background leakage and improved sensitivity. Furthermore, the FAC-HCR was used to test a non-nucleic acid target, apurinic/apyrimidinic endonuclease 1 (APE1), an important BER-involved endonuclease. The fluorescence analysis results confirmed that FAC-HCR can reach a detection limit of 0.1174 U/mL. By using the two HPs for FAC-HCR with polyetherimide-based nanoparticles, the activity of APE1 in living cells can be imaged. In summary, this study could provide a new idea to design an FA-based HCR and improve the performance of HCRs in live cell imaging.