Fine-tuning of stable organic free-radical photosensitizers for photodynamic immunotherapy.

Photodynamic immunotherapy (PDI) is an innovative approach to cancer treatment that utilizes photodynamic therapy (PDT) and photosensitizers (PSs) to induce immunogenic cell death (ICD). However, currently most commonly used PSs have restricted capabilities to generate reactive oxygen species (ROS) via a type-II mechanism under hypoxic environments, which limits their effectiveness in PDI. To overcome this, we propose a novel approach for constructing oxygen independent PSs based on stable organic free-radical molecules. By fine-tuning the characteristics of tris(2,4,6-trichlorophenyl)-methyl (TTM) radicals through the incorporation of electron-donating moieties, we successfully found that TTMIndoOMe could produce substantial amounts of ROS even in hypoxic environments. In vitro experiments showed that TTMIndoOMe could effectively produce O2˙-, kill tumor cells and trigger ICD. Moreover, in vivo experiments also demonstrated that TTMIndoOMe could further trigger anti-tumor immune response and exhibit a superior therapeutic effect compared with PDT alone. Our study offers a promising approach towards the development of next-generation PSs functioning efficiently even under hypoxic conditions and also paves the way for the creation of more effective PSs for PDI.

Cross-Modal Retrieval Between 13C NMR Spectra and Structures Based on Focused Libraries.

Library matching by comparing carbon-13 nuclear magnetic resonance (13C NMR) spectra with spectral data in the library is a crucial method for compound identification. In our previous paper, we introduced a deep contrastive learning system called CReSS, which used a library that contained more structures. However, CReSS has two limitations: there were no unknown structures in the library, and a redundant library reduces the structure-elucidation accuracy. Herein, we replaced the oversize traditional libraries with focused libraries containing a small number of molecules. A previously generative model, CMGNet, was used to generate focused libraries for CReSS. The combined model achieved a Top-10 accuracy of 54.03% when tested on 6,471 13C NMR spectra. In comparison, CReSS with a random reference structure library achieved an accuracy of only 9.17%. Furthermore, to expand the advantages of the focused libraries, we proposed SAmpRNN, which is a recurrent neural network (RNN). With the large focused library amplified by SAmpRNN, the structure-identification accuracy of the model increased in 70.0% of the 30 random example cases. In general, cross-modal retrieval between 13C NMR spectra and structures based on focused libraries (CFLS) achieved high accuracy and provided more accurate candidate structures than traditional libraries for compound identification.

DOTA-Based Plectin-1 Targeted Contrast Agent Enables Detection of Pancreatic Cancer in Human Tissue.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy with extremely poor patient survival rates. A key reason for the poor prognosis is the lack of effective diagnostic tools to detect the disease at curable, premetastatic stages. Tumor surgical resection is PDAC's first-line treatment, however distinguishing between cancerous and healthy tissue with current imaging tools remains a challenge. In this work, we report a DOTA-based fluorescent probe targeting plectin-1 for imaging PDAC with high specificity. To enable heterogeneous functionalization of the DOTA-core with multiple targeting peptide units and the fluorophore, a novel, fully clickable synthetic route that proceeds in one pot was developed. Extensive validation of the probe set the stage for PDAC detection in mice and human tissue. Altogether, these findings may pave the way for improved clinical understanding and early detection of PDAC progression as well as more accurate resection criteria.

Unravelling Immune-Inflammatory Responses and Lysosomal Adaptation: Insights from Two-Photon Excited Delayed Fluorescence Imaging.

Two-photon excitation (TPE) microscopy with near-infrared (NIR) emission has emerged as a promising technique for deep-tissue optical imaging. Recent developments in fluorescence lifetime imaging with long-lived emission probes have further enhanced the spatial resolution and precision of fluorescence imaging, especially in complex systems with short-lived background signals. In this study, two innovative lysosome-targeting probes, Cz-NA and tCz-NA, are introduced. These probes offer a combination of advantages, including TPE (λex = 880 nm), NIR emission (λem = 650 nm), and thermally activated delayed fluorescence (TADF) with long-lived lifetimes (1.05 and 1.71 µs, respectively). These characteristics significantly improve the resolution and signal-to-noise ratio in deep-tissue imaging. By integrating an acousto-optic modulator (AOM) device with TPE microscopy, the authors successfully applied Cz-NA in two-photon excited delayed fluorescence (TPEDF) imaging to track lysosomal adaptation and immune responses to inflammation in mice. This study sheds light on the relationship between lysosome tubulation, innate immune responses, and inflammation in vivo, providing valuable insights for the development of autofluorescence-free molecular probes in the future.

In Situ Photoacoustic Visualization of Pneumonia Induced by MRSA and Specific Identifying Tumor-Homing Bacteria.

Optical imaging holds great promise for monitoring bacterial infectious processes and drug resistance with high temporal-spatial resolution. Currently, the diagnosis of deep-seated bacterial infections in vivo with fluorescence imaging, including near-infrared (NIR) fluorescence imaging technology, remains a significant challenge due to its limited tissue penetration depth. In this study, we developed a highly specific targeting probe, Cy7-Neo-NO2, by conjugating a bacterial 16S rRNA-targeted moiety, neomycin, with a bacterial nitroreductase (NTR)-activated NIR photoacoustic (PA) scaffold using our previously developed caged photoinduced electron transfer (a-PeT) approach. This conjugation effectively resolved probe aggregation issues in physiological conditions and substantially enhanced its reactivity toward bacterial NTR. Notably, Cy7-Neo-NO2 enabled the first in situ photoacoustic imaging of pneumonia induced by methicillin-resistant Staphylococcus aureus (MRSA), as well as the detection of bacteria within tumors. Furthermore, upon NIR irradiation, Cy7-Neo-NO2 successfully inhibited MRSA growth through a synergistic effect combining photothermal therapy and photodynamic therapy. Our results provided an effective tool for obtaining exceptional PA agents for accurate diagnosis, therapeutic evaluation of deep-seated bacterial infections in vivo, and intratumoral bacteria-specific recognition.

Advances in the Application of Artificial Intelligence-Based Spectral Data Interpretation: A Perspective.

The interpretation of spectral data, including mass, nuclear magnetic resonance, infrared, and ultraviolet-visible spectra, is critical for obtaining molecular structural information. The development of advanced sensing technology has multiplied the amount of available spectral data. Chemical experts must use basic principles corresponding to the spectral information generated by molecular fragments and functional groups. This is a time-consuming process that requires a solid professional knowledge base. In recent years, the rapid development of computer science and its applications in cheminformatics and the emergence of computer-aided expert systems have greatly reduced the difficulty in analyzing large quantities of data. For expert systems, however, the problem-solving strategy must be known in advance or extracted by human experts and translated into algorithms. Gratifyingly, the development of artificial intelligence (AI) methods has shown great promise for solving such problems. Traditional algorithms, including the latest neural network algorithms, have shown great potential for both extracting useful information and processing massive quantities of data. This Perspective highlights recent innovations covering all of the emerging AI-based spectral interpretation techniques. In addition, the main limitations and current obstacles are presented, and the corresponding directions for further research are proposed. Moreover, this Perspective gives the authors' personal outlook on the development and future applications of spectral interpretation.

Polymyxin-based fluorescent probes to combat Gram-negative antimicrobial resistance.

Reliable diagnostic approaches especially those targeting critical Gram-negative bacteria are urgently needed for the prevention of antimicrobial resistance. Polymyxin B (PMB) which specifically targets the outer membrane of Gram-negative bacteria is the last-line antibiotic against life-threatening multidrug-resistant Gram-negative bacteria. However, increasing number of studies have reported the spread of PMB-resistant strains. With the aim to specifically detect Gram-negative bacteria and potentially reduce the irrational use of antibiotics, we herein rationally designed two Gram-negative bacteria specific fluorescent probes based on our previous activity-toxicity optimization of PMB. The in vitro probe PMS-Dns showed fast and selective labeling of Gram-negative pathogens in complex biological cultures. Subsequently, we constructed the caged in vivo fluorescent probe PMS-Cy-NO2 by conjugating bacterial nitroreductase (NTR)-activatable positive charged hydrophobic near-infrared (NIR) fluorophore with polymyxin scaffold. Significantly, PMS-Cy-NO2 exhibited excellent Gram-negative bacterial detection capability with the differentiation between Gram-positive and Gram-negative in a mouse skin infection model.

Analyte-Triggered Excited-State Intramolecular Proton Transfer- Delayed Fluorescence: A General Approach for Time-Resolved Turn-On Fluorescence Imaging.

The research of delayed fluorescence (DF) has been a hot topic in biological imaging. However, the development of analyte-triggered small molecule DF probes remains a considerable challenge. Herein a novel excited-state intramolecular proton transfer-delayed fluorescence (ESIPT-DF) approach to construct analyte-stimulated DF probes was reported. These new classes of ESIPT-DF luminophores were strategically designed and synthesized by incorporating 2-(2'-hydroxyphenyl)benzothiazole (HBT), a known ESIPT-based fluorophore, as acceptor with a series of classic donor moieties, which formed a correspondingly twisted donor-acceptor pair within each molecule. Thereinto, HBT-PXZ and HBT-PTZ exhibited significant ESIPT and DF characters with lifetimes of 5.37 and 3.65 µs in the solid state, respectively. Furthermore, a caged probe HBT-PXZ-Ga was developed by introducing a hydrophilic d-galactose group as the recognition unit specific for ß-galactosidase (ß-gal) and ESIPT-DF blocking agent and applied to investigate the influence of metal ions on ß-gal activity on the surface of Streptococcus pneumoniae as a convenient tool. This ESIPT-DF "turn-on" approach is easily adaptable for the measurement of many different analytes using only a predictable modification on the caged group without modification of the core structure.

Membrane dual-targeting probes: A promising strategy for fluorescence-guided prostate cancer surgery and lymph node metastases detection.

Fluorescence-guided surgery (FGS) with tumor-targeted imaging agents, particularly those using the near-infrared wavelength, has emerged as a real-time technique to highlight the tumor location and margins during a surgical procedure. For accurate visualization of prostate cancer (PCa) boundary and lymphatic metastasis, we developed a new approach involving an efficient self-quenched near-infrared fluorescence probe, Cy-KUE-OA, with dual PCa-membrane affinity. Cy-KUE-OA specifically targeted the prostate-specific membrane antigen (PSMA), anchored into the phospholipids of the cell membrane of PCa cells and consequently showed a strong Cy7-de-quenching effect. This dual-membrane-targeting probe allowed us to detect PSMA-expressing PCa cells both in vitro and in vivo and enabled clear visualization of the tumor boundary during fluorescence-guided laparoscopic surgery in PCa mouse models. Furthermore, the high PCa preference of Cy-KUE-OA was confirmed on surgically resected patient specimens of healthy tissues, PCa, and lymph node metastases. Taken together, our results serve as a bridge between preclinical and clinical research in FGS of PCa and lay a solid foundation for further clinical research.

Characterization of Chlorogenic Acid as a Two-Photon Fluorogenic Probe that Regulates Glycolysis in Tumor Cells under Hypoxia.

High levels of steady-state mitochondrial reactive oxygen species (ROS) and glycolysis are hallmarks of cancer. An improved understanding of interactions between tumor energetics and mitochondrial ROS modulation is useful for the development of new anticancer strategies. Here, we show that the natural product chlorogenic acid (CGA) specifically scavenged abnormally elevated mitochondrial O2•- and exhibited a two-photon fluorescence turn-on response to tumor cells under hypoxia and tumor tissues in vivo. Furthermore, we illustrated that CGA treatment reduced O2•- levels in cells, hampered activation of AMP-activated protein kinase (AMPK), and shifted metabolism from glycolysis to oxidative phosphorylation (OXPHOS), resulting in inhibition of tumor growth under hypoxia. This study demonstrates an efficient two-photon fluorescent tool for real-time assessment of mitochondrial O2•- and a clear link between reducing intracellular ROS levels by CGA treatments and regulating metabolism, as well as undeniably helpful insights for the development of new anticancer strategies.

Thermally activated delayed fluorescence emitters: a thionation approach toward next-generation photosensitizers.

Achieving highly efficient intersystem crossing (ISC) remains a key focus in the design of heavy atom-free photosensitizers (PSs) for various photophysical and photochemical applications. Herein, we report a general and robust molecular design strategy for obtaining photoactivatable heavy atom-free PSs by performing a simple sulfur substitution of carbonyl oxygen atoms of a thermally activated delayed fluorescence (TADF) emitter. This thionation led to a significant fluorescence loss, resulting in an increased ISC transformation. Upon white-light irradiation, the sulfur-substituted TADF compound (S-AIOH-Cz) exhibited a long-lived fluorescence turn-on response, a long-lasting triplet state lifetime and a superior reactive oxygen species (ROS) generation ability, which is desirable for time-resolved fluorescence imaging and photodynamic disinfection against antimicrobial resistance.

Hyperglycemia in Diabetic Skin Infections Promotes Staphylococcus aureus Virulence Factor Aureolysin: Visualization by Molecular Imaging.

Bacterial skin infections are common in diabetic patients, with Staphylococcus aureus (S. aureus) being the most commonly isolated, causing comorbidities such as increased mortality and long-term hospitalization. While precise mechanisms remain to be determined, hyperglycemia represents an important pathogenetic factor responsible for the increased risk of S. aureus infection. Herein, we constructed a series of ratiometric fluorescent molecular probes for aureolysin (Aur), a major virulence factor in S. aureus. Using probe 1, we were able to determine specific Aur activity in both cells and tissues. We also observed that elevated glucose levels led to 2-fold higher Aur expression in S. aureus cultures. In a diabetic mouse model, we used molecular imaging to demonstrate that hyperglycemia tripled S. aureus Aur virulence compared to nondiabetic mice, resulting in more severe infections.

Donor manipulation for constructing a pH sensing thermally activated delayed fluorescent probe to detect alkaliphiles.

pH homeostasis is essential for alkaliphiles, given their widespread use in biotechnological applications. However, quantitative monitoring of alkaline pH in alkaliphiles remains challenging. Here, we synthesized for the first time, a thermally activated delayed fluorescent (TADF) pH probe: NI-D-OH. Our probe exhibits a good linear relationship between fluorescence intensity and pH in the neutral to alkaline range (pH 7.0-8.6), as well as long-lived TADF emission. We further show that NI-D-OH can be used to monitor intracellular pH in living organisms, and evaluate the effect of Na+ on the pH homeostasis, demonstrating the potential for alkaline pH monitoring and time-resolved fluorescence imaging.

Developing fluoromodule-based probes for in vivo monitoring the bacterial infections and antibiotic responses.

Recently, antibiotic resistant has become a serious public health concern, which warrants new generations of antibiotics to be developed. Pharmacodynamic evaluation is crucial in drug discovery processes. Despite numerous advanced imaging systems are available nowadays, technologies for the sensitive in vivo diagnosis of bacterial infections and direct visualization of drug efficacy are yet to be developed. In this study, we have developed novel near-infrared (NIR) fluorogenic probes. These probes are dark in solution but highly fluorescent when bound to the cognate reporter, fluorogen-activating protein (FAP). We established the in vivo bacterial infection model using FAP_dH6.2 recombinantly expressed E. coli and applied this NIR fluoromodule-based system for diagnosing bacterial infections and monitoring disease progressions and its responses to a type of antibiotics through classic mechanism of membrane lysis. This NIR fluoromodule-based system will discover new information on bacterial infections and identify newer antibacterial entities.

DOTAM-Based, Targeted, Activatable Fluorescent Probes for the Highly Sensitive and Selective Detection of Cancer Cells.

The utilization of an activatable, substrate-based probe design in combination with a cellular targeting approach has been rarely explored for cancer imaging on a small-molecule basis, although such probes could benefit from advantages of both concepts. Cysteine proteases like cathepsin S are known to be involved in fundamental processes associated with tumor development and progression and thus are valuable cancer markers. We report the development of a combined dual functional DOTAM-based, RGD-targeted internally quenched fluorescent probe that is activated by cathepsin S. The probe exhibits excellent in vitro activation kinetics which can be fully translated to human cancer cell lines. We demonstrate that the targeted, activatable probe is superior to its nontargeted analog, exhibiting improved uptake into ανß3-integrin expressing human sarcoma cells (HT1080) and significantly higher resultant fluorescence staining. However, profound activation was also found in cancer cells with a lower integrin expression level, whereas in healthy cells almost no probe activation could be observed, highlighting the high selectivity of our probe toward cancer cells. These auspicious results show the outstanding potential of the dual functionality concept combining a substrate-based probe design with a targeting approach, which could form the basis for highly sensitive and selective in vivo imaging probes.

Dual-Mode Detection of Bacterial 16S Ribosomal RNA in Tissues.

The specific detection of pathogens has long been recognized as a vital strategy for controlling bacterial infections. Herein, a novel hydrophilic aromatic-imide-based thermally activated delayed fluorescence (TADF) probe, AI-Cz-Neo, is designed and synthesized by the conjugation of a TADF emitter with a bacterial 16S ribosomal RNA-targeted moiety, neomycin. Biological data showed for the first time that AI-Cz-Neo could be successfully applied for the dual-mode detection of bacterial 16S rRNA using confocal fluorescence imaging and time-resolved fluorescence imaging (TRFI) in both cells and tissues. These findings greatly expand the application of TADF fluorophores in time-resolved biological imaging and provide a promising strategy for the precise and reliable diagnosis of bacterial infections based on the dual-mode imaging of bacterial 16S rRNA by fluorescence intensity and fluorescence lifetime.

An Activatable Lanthanide Luminescent Probe for Time-Gated Detection of Nitroreductase in Live Bacteria.

Herein we report the development of a turn-on lanthanide luminescent probe for time-gated detection of nitroreductases (NTRs) in live bacteria. The probe is activated through NTR-induced formation of the sensitizing carbostyril antenna and resulting energy transfer to the lanthanide center. This novel NTR-responsive trigger is virtually non-fluorescent in its inactivated form and features a large signal increase upon activation. We show that the probe is capable of selectively sensing NTR in lysates as well as in live bacteria of the ESKAPE family which are clinically highly relevant multiresistant pathogens responsible for the majority of hospital infections. The results suggest that our probe could be used to develop diagnostic tools for bacterial infections.

A single component self-assembled thermally activated delayed fluorescence nanoprobe.

A novel versatile thermally activated delayed fluorescence (TADF) nanoprobe, AI-Cz-NP, was designed and fabricated through self-assembly of a single-component amphiphilic monomer for potential applications in confocal imaging and time-resolved fluorescence imaging.

Rapid differentiation between bacterial infections and cancer using a near-infrared fluorogenic probe.

The reliable differentiation between bacterial infections and other pathologies is crucial for both diagnostics and therapeutic approaches. To accommodate such needs, we herein report the development of an activatable near-infrared fluorescent probe 1 that could be applied in the ultrafast, ultrasensitive and specific detection of nitroreductase (NTR) activity in bacterial pathogens both in vitro and in vivo. Upon reaction with NTR, the nitro-group of the para-nitro phenyl sulfonic moiety present in probe 1 was reduced to an amino-group, resulting in a near-infrared fluorescence turn-on of the latent cyanine 7 fluorophore. Probe 1 was capable of rapid and real-time quantitative detection of 0-150 ng mL-1 NTR with a limit of detection as low as 0.67 ng mL-1 in vitro. In addition, probe 1 exhibited an outstanding performance of ultrafast measurements and suitable selectivity toward NTR to accurately sense intracellular basal NTR in ESKAPE bacterial pathogens. Most remarkably, probe 1 was capable of noninvasively identifying bacterial infection sites without showing any significantly increased signal of tumour sites in the same animal within 30 min.

Solanrubiellin A, a hydroanthraquinone dimer with antibacterial and cytotoxic activity from Solanum lyratum.

A new hydroanthraquinone dimer derivative, solanrubiellin A, was isolated from the whole plants of Solanum lyratum. The structure of 1 was established through extensive NMR spectroscopy analysis, and the absolute configuration was elucidated by comparison of its experimental and calculated ECD spectra. Compound 1 showed antibacterial activity with MIC values of 2-10 µM against several Gram-positive bacteria. Compound 1 also demonstrated cytotoxic activity against human A549, HT-29 and HL-60 cell lines with IC50 values ranging from 2.06 to 9.35 µM.