Specific Detection and Imaging of Enzyme Activity by Signal‐Amplifiable Self‐Assembling 19F MRI Probes

Specific turn‐on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal‐amplifiable self‐assembling ¹⁹F NMR/MRI probe for turn‐on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are “silent” when aggregated, but exhibit a disassembly driven turn‐on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these ¹⁹F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by ¹⁹F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by ¹⁹F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn‐on ¹⁹F probes may serve as a screening platform for the activity of MMPs.     

Development of Zinc‐Specific iCEST MRI as an Imaging Biomarker for Prostate Cancer

The healthy prostate contains the highest concentration of mobile zinc in the body. As this level decreases dramatically during the initial development of prostate cancer, in vivo detection of prostate zinc content may be applied for diagnosis of prostate cancer. Using ¹⁹F ion chemical exchange saturation transfer magnetic resonance imaging (iCEST MRI) and TF‐BAPTA as a fluorinated Zn‐binding probe with micromolar sensitivity, we show that iCEST MRI is able to differentiate between normal and malignant prostate cells with a 10‐fold difference in contrast following glucose‐stimulated zinc secretion in vitro. The iCEST signal decreased in normal prostate cells upon downregulation of the ZIP1 zinc transporter. In vivo, using an orthotopic prostate cancer mouse model and a transgenic adenocarcinoma of the mouse prostate (TRAMP) model, a gradual decrease of >300 % in iCEST contrast following the transition of normal prostate epithelial cells to cancer cells was detected.     

Cell Tracking with Caged Xenon: Using Cryptophanes as MRI Reporters upon Cellular Internalization

Caged xenon has great potential in overcoming sensitivity limitations for solution‐state NMR detection of dilute molecules. However, no application of such a system as a magnetic resonance imaging (MRI) contrast agent has yet been performed with live cells. We demonstrate MRI localization of cells labeled with caged xenon in a packed‐bed bioreactor working under perfusion with hyperpolarized‐xenon‐saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell‐associated versus unbound cages. We present MR images with 10³‐fold sensitivity enhancement for cell‐internalized, dual‐mode (fluorescence/MRI) xenon hosts at low micromolar concentrations. Our results illustrate the capability of functionalized xenon to act as a highly sensitive cell tracer for MRI detection even without signal averaging. The method will bridge the challenging gap for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications.     

Synthesis of Intrinsically Disordered Fluorinated Peptides for Modular Design of High-Signal 19F MRI Agents

¹⁹F MRI is valuable for in vivo imaging due to the only trace amounts of fluorine in biological systems. Because of the low sensitivity of MRI however, designing new fluorochemicals remains a significant challenge for achieving sufficient ¹⁹F signal. Here, we describe a new class of high-signal, water-soluble fluorochemicals as ¹⁹F MRI imaging agents. A polyamide backbone is used for tuning the proteolytic stability to avoid retention within the body, which is a limitation of current state-of-the-art perfluorochemicals. We show that unstructured peptides containing alternating N-ϵ-trifluoroacetyllysine and lysine provide a degenerate ¹⁹F NMR signal. ¹⁹F MRI phantom images provide sufficient contrast at micromolar concentrations, showing promise for eventual clinical applications. Finally, the degenerate high signal characteristics were retained when conjugated to a large protein, indicating potential for in vivo targeting applications, including molecular imaging and cell tracking.     

Glycan Metabolic Fluorine Labeling for In Vivo Visualization of Tumor Cells and In Situ Assessment of Glycosylation Variations

The abnormality in the glycosylation of surface proteins is critical for the growth and metastasis of tumors and their capacity for immunosuppression and drug resistance. This anomaly offers an entry point for real-time analysis on glycosylation fluctuations. In this study, we report a strategy, glycan metabolic fluorine labeling (MEFLA), for selectively tagging glycans of tumor cells. As a proof of concept, we synthesized two fluorinated unnatural monosaccharides with distinctive ¹⁹F chemical shifts (Ac₄ManNTfe and Ac₄GalNTfa). These two probes could undergo selective uptake by tumor cells and subsequent incorporation into surface glycans. This approach enables efficient and specific ¹⁹F labeling of tumor cells, which permits in vivo tracking of tumor cells and in situ assessment of glycosylation changes by ¹⁹F MRI. The efficiency and specificity of our probes for labeling tumor cells were verified in vitro with A549 cells. The feasibility of our method was further validated with in vivo experiments on A549 tumor-bearing mice. Moreover, the capacity of our approach for assessing glycosylation changes of tumor cells was illustrated both in vitro and in vivo. Our studies provide a promising means for visualizing tumor cells in vivo and assessing their glycosylation variations in situ through targeted multiplexed ¹⁹F MRI.     

Carbon Dots as a New Class of Diamagnetic Chemical Exchange Saturation Transfer (diaCEST) MRI Contrast Agents

While carbon dots (C‐dots) have been extensively investigated pertaining to their fluorescent, phosphorescent, electrochemiluminescent, optoelectronic, and catalytic features, their inherent chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) properties are unknown. By virtue of their hydrophilicity and abundant exchangeable protons of hydroxyl, amine, and amide anchored on the surface, we report here that C‐dots can be adapted as effective diamagnetic CEST (diaCEST) MRI contrast agents. As a proof‐of‐concept demonstration, human glioma cells were labeled with liposomes with or without encapsulated C‐dots and implanted in mouse brain. In vivo CEST MRI was able to clearly differentiate labeled cells from non‐labeled cells. The present findings may encourage new applications of C‐dots for in vivo imaging in deep tissues, which is currently not possible using conventional fluorescent (near‐infrared) C‐dots.     

Mesoporous silica nanoparticles for 19F magnetic resonance imaging,fluorescence imaging,and drug delivery

Multifunctional mesoporous silica nanoparticles (MSNs) are good candidates for multimodal applications in drug delivery, bioimaging, and cell targeting. In particular, controlled release of drugs from MSN pores constitutes one of the superior features of MSNs. In this study, a novel drug delivery carrier based on MSNs, which encapsulated highly sensitive ¹⁹F magnetic resonance imaging (MRI) contrast agents inside MSNs, was developed. The nanoparticles were labeled with fluorescent dyes and functionalized with small molecule-based ligands for active targeting. This drug delivery system facilitated the monitoring of the biodistribution of the drug carrier by dual modal imaging (NIR/¹⁹F MRI). Furthermore, we demonstrated targeted drug delivery and cellular imaging by the conjugation of nanoparticles with folic acid. An anticancer drug (doxorubicin, DOX) was loaded in the pores of folate-functionalized MSNs for intracellular drug delivery. The release rates of DOX from the nanoparticles increased under acidic conditions, and were favorable for controlled drug release to cancer cells. Our results suggested that MSNs may serve as promising ¹⁹F MRI-traceable drug carriers for application in cancer therapy and bio-imaging.     

FSiNPs mediated improved double immunofluorescence staining for gastric cancer cells imaging

A fluorescent silica nanoparticles (FSiNPs) mediated double immunofluorescence staining technique has been proposed for MGC-803 gastric cancer cells imaging by confocal laser scanning microscopy. Anti-CEA antibody and anti-CK19 antibody which can be both bonded to MGC-803 gastric cancer cells were first conjugated to fluorescein isothiocyanate (FITC) doped fluorescent silica nanoparticles (FFSiNPs) and RuBPY doped fluorescent silica nanoparticles (RFSiNPs), respectively. The MGC-803 gastric cancer cells were incubated with the mixture of anti-CEA antibody-conjugated FFSiNPs and anti-CK19 antibody-conjugated RFSiNPs, and subsequently imaged using confocal laser scanning microscopy. With this method, the in vitro cultured MGC-803 gastric cancer cells lines were successfully doubled labeled and distinguished through antigen-antibody recognition, together with the green and red signal of FFSiNPs and RFSiNPs simultaneously obtained without crossreactivity by confocal laser scanning microscopy imaging. By comparison with the conventional double immunofluorescence staining using green-emitting and red-emitting dyes, the photostability of this proposed method for confocal laser scanning microscopy imaging has been greatly improved. Furthermore, the ex vivo imaging of primary MGC-803 gastric cancer cells samples came from the tumor tissues of mice bearing the MGC gastric cancer tumor xenografts by this method have also been explored. The results demonstrate that the method offers potential advantage of photostability for the confocal laser scanning microscopy imaging of MGC-803 gastric cancer cells, and is applicable to the imaging of primary MGC-803 gastric cancer cells from the tumor tissues.     

In vivo magnetic resonance detection of cancer by using multifunctional magnetic nanocrystals

The unique properties of magnetic nanocrystals provide them with high potential as key probes and vectors in the next generation of biomedical applications. Although superparamagnetic iron oxide nanocrystals have been extensively studied as excellent magnetic resonance imaging (MRI) probes for various cell trafficking, gene expression, and cancer diagnosis, further development of in vivo MRI applications has been very limited. Here, we describe in vivo diagnosis of cancer, utilizing a well-defined magnetic nanocrystal probe system with multiple capabilities, such as small size, strong magnetism, high biocompatibility, and the possession of active functionality for desired receptors. Our magnetic nanocrystals are conjugated to a cancer-targeting antibody, Herceptin, and subsequent utilization of these conjugates as MRI probes has been successfully demonstrated for the monitoring of in vivo selective targeting events of human cancer cells implanted in live mice. Further conjugation of these nanocrystal probes with fluorescent dye-labeled antibodies enables both in vitro and ex vivo optical detection of cancer as well as in vivo MRI, which are potentially applicable for an advanced multimodal detection system. Our study finds that high performance in vivo MR diagnosis of cancer is achievable by utilizing improved and multifunctional material properties of iron oxide nanocrystal probes.     

Systematic study of protein detection mechanism of self-assembling 19F NMR/MRI nanoprobes toward rational design and improved sensitivity

(19)F NMR/MRI probe is expected to be a powerful tool for selective sensing of biologically active agents owing to its high sensitivity and no background signals in live bodies. We have recently reported a unique supramolecular strategy for specific protein detection using a protein ligand-tethered self-assembling (19)F probe. This method is based on a recognition-driven disassembly of the nanoprobes, which induced a clear turn-on signal of (19)F NMR/MRI. In the present study, we conducted a systematic investigation of the relationship between structure and properties of the probe to elucidate the mechanism of this turn-on (19)F NMR sensing in detail. Newly synthesized (19)F probes showed three distinct behaviors in response to the target protein: off/on, always-on, and always-off modes. We clearly demonstrated that these differences in protein response could be explained by differences in the stability of the probe aggregates and that "moderate stability" of the aggregates produced an ideal turn-on response in protein detection. We also successfully controlled the aggregate stability by changing the hydrophobicity/hydrophilicity balance of the probes. The detailed understanding of the detection mechanism allowed us to rationally design a turn-on (19)F NMR probe with improved sensitivity, giving a higher image intensity for the target protein in (19)F MRI.     

A New Fluorescent Probe for Selective Cd2+ Detection and Cell Imaging

A new highly selective fluorescent probe, (E)‐4‐(4‐([2,2′:6′,2′′‐terpyridin]‐4′‐yl)styryl)‐1‐octadecylpyridin‐1‐ium bromide (ZC‐F8), was designed and synthesized for cadmium detection and cell imaging. The fluorescence spectra of ZC‐F8 exhibited its excellent response towards Cd²⁺ via intramolecular charge transfer effect and aggregation induced emission effect. The cell‐imaging experiment was carried out to estimate the actual biological application of ZC‐F8. The probe displayed ideal membrane permeable and labeled property for cadmium, indicating its promising application for metal ions detection and tracing in living cells.     

Manganese-Zinc Ferrites: Safe and Efficient Nanolabels for Cell Imaging and Tracking In Vivo

Manganese-zinc ferrite nanoparticles were synthesized by using a hydrothermal treatment, coated with silica, and then tested as efficient cellular labels for cell tracking, using magnetic resonance imaging (MRI) in vivo. A toxicity study was performed on rat mesenchymal stem cells and C6 glioblastoma cells. Adverse effects on viability and cell proliferation were observed at the highest concentration (0.55 mM) only; cell viability was not compromised at lower concentrations. Nanoparticle internalization was confirmed by transmission electron microscopy. The particles were found in membranous vesicles inside the cytoplasm. Although the metal content (0.42 pg Fe/cell) was lower compared to commercially available iron oxide nanoparticles, labeled cells reached a comparable relaxation rate R₂, owing to higher nanoparticle relaxivity. Cells from transgenic luciferase-positive rats were used for in vivo experiments. Labeled cells were transplanted into the muscles of non-bioluminescent rats and visualized by MRI. The cells produced a distinct hypointense signal in T₂- or T₂*-weighted MR images in vivo. Cell viability in vivo was verified by bioluminescence.     

Slow exchange in the chromophore of a green fluorescent protein variant

Green fluorescent protein and its mutants have become valuable tools in molecular biology. They also provide systems rich in photophysical and photochemical phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. Here, we report the (19)F nuclear magnetic resonance (NMR) studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow molecular motions in these proteins. The concerted use of dynamic NMR and (19)F relaxation measurements, supported by temperature, concentration- and folding-dependent experiments provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. (19)F NMR relaxation and line shape analysis indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodynamic analysis revealed a difference in enthalpy (Delta)H(0) = (18.2 +/- 3.8) kJ/mol and entropy T(Delta)S(0) = (19.6 +/- 1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estimated to be approximately 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("atomic mutations") like H --> F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

Live‐cell MRI with Xenon Hyper‐CEST Biosensors Targeted to Metabolically Labeled Cell‐Surface Glycans

The targeting of metabolically labeled glycans with conventional MRI contrast agents has proved elusive. In this work, which further expands the utility of xenon Hyper‐CEST biosensors in cell experiments, we present the first successful molecular imaging of such glycans using MRI. Xenon Hyper‐CEST biosensors are a novel class of MRI contrast agents with very high sensitivity. We designed a multimodal biosensor for both fluorescent and xenon MRI detection that is targeted to metabolically labeled sialic acid through bioorthogonal chemistry. Through the use of a state of the art live‐cell bioreactor, it was demonstrated that xenon MRI biosensors can be used to image cell‐surface glycans at nanomolar concentrations.     

Preparation of glutathione loaded nanoemulsions and testing of hepatoprotective activity on THLE-2 cells

To improve bioavailability and stability of hydrophobic and hydrophilic compounds, nanoemulsions are good alternatives as delivery systems because of their nontoxic and nonirritant nature. Glutathione (GSH) suffers from low stability in water, where its encapsulation in nanoemulsions is a powerful strategy to its stability in aqueous systems. The aim of this study was to obtain nanoemulsions from the hydrophobic/hydrophilic contents of N. sativa seed oil so as to improve GSH stability along with bioavailability of N. sativa seed oil. Then, the prepared nanoemulsions were tested for in vitro hepatoprotective activity against ethanol toxicity. To the best of our knowledge, there is no study on the test of nanoemulsions by the combination of Nigella sativa seed oils and GSH in hepatoprotective activity. Here, nanoemulsions with different contents were prepared using Nigella sativa seed oils. Content analyses and characterisation studies of prepared nanoemulsions were carried out. In order to investigate the protective effects against to ethanol exposure, THLE-2 cells were pretreated with nanoemulsions for 2 h with the maximum benign dose (0.5 mg/mL of nanoemulsions). Ethanol (400 mM) was introduced to pretreated cells and nontreated cells for 48- or 72-h periods, followed by cell viability assay was carried out. Fluorescence microscopy tests revealed the introduction of the nanoemulsions into THLE-2 cells. The findings show that nanoformulations have promising in vitro hepatoprotective effects on the THLE-2 cell line against ethanol exposure.     

Elastic Polymer Coated Nanoparticles with Fast Clearance for 19F MR Imaging

¹⁹F magnetic resonance imaging (MRI) is a powerful molecular imaging technique that enables high-resolution imaging of deep tissues without background signal interference. However, the use of nanoparticles (NPs) as ¹⁹F MRI probes has been limited by the immediate trapping and accumulation of stiff NPs, typically of around 100 nm in size, in the mononuclear phagocyte system, particularly in the liver. To address this issue, elastic nanomaterials have emerged as promising candidates for improving delivery efficacy in vivo. Nevertheless, the impact of elasticity on NP elimination has remained unclear due to the lack of suitable probes for real-time and long-term monitoring. In this study, we present the development of perfluorocarbon-encapsulated polymer NPs as a novel ¹⁹F MRI contrast agent, with the aim of suppressing long-term accumulation. The polymer NPs have high elasticity and exhibit robust sensitivity in ¹⁹F MRI imaging. Importantly, our ¹⁹F MRI data demonstrate a gradual decline in the signal intensity of the polymer NPs after administration, which contrasts starkly with the behavior observed for stiff silica NPs. This innovative polymer-coated NP system represents a groundbreaking nanomaterial that successfully overcomes the challenges associated with long-term accumulation, while enabling tracking of biodistribution over extended periods.     

Enzymatically Shifting Nitroxides for EPR Spectroscopy and Overhauser‐Enhanced Magnetic Resonance Imaging

In vivo investigations of enzymatic processes using non‐invasive approaches are a long‐lasting challenge. Recently, we showed that Overhauser‐enhanced MRI is suitable to such a purpose. A β‐phosphorylated nitroxide substrate prototype exhibiting keto–enol equilibrium upon enzymatic activity has been prepared. Upon enzymatic hydrolysis, a large variation of the phosphorus hyperfine coupling constant (Δa_P=4 G) was observed. The enzymatic activities of several enzymes were conveniently monitored by electronic paramagnetic resonance (EPR). Using a 0.2 T MRI machine, in vitro and in vivo OMRI experiments were successfully performed, affording a 1200 % enhanced MRI signal in vitro, and a 600 % enhanced signal in vivo. These results highlight the enhanced imaging potential of these nitroxides upon specific enzymatic substrate‐to‐product conversion.     

Multifunctional Core–Shell Silica Nanoparticles for Highly Sensitive 19F Magnetic Resonance Imaging

¹⁹F magnetic resonance imaging (¹⁹F MRI) is useful for monitoring particular signals from biological samples, cells, and target tissues, because background signals are missing in animal bodies. Therefore, highly sensitive ¹⁹F MRI contrast agents are in great demand for their practical applications. However, we have faced the following challenges: 1) increasing the number of fluorine atoms decreases the solubility of the molecular probes, and 2) the restriction of the molecular mobility attenuates the ¹⁹F MRI signals. Herein, we developed novel multifunctional core–shell nanoparticles to solve these issues. They are composed of a core micelle filled with liquid perfluorocarbon and a robust silica shell. These core–shell nanoparticles have superior properties such as high sensitivity, modifiability of the surface, biocompatibility, and sufficient in vivo stability. By the adequate surface modifications, gene expression in living cells and tumor tissue in living mice were successfully detected by ¹⁹F MRI.