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.     

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.     

Functional Hybrid Molecules for the Visualization of Cancer: PESIN-Homodimers Combined with Multimodal Molecular Imaging Probes for Positron Emission Tomography and Optical Imaging: Suited for Tracking of GRPR-Positive Malignant Tissue**

We describe multimodal imaging probes for gastrin-releasing peptide receptor (GRPR)-specific targeting suited for positron emission tomography and optical imaging (PET/OI), consisting of PESIN (PEG₃-BBN_7-14) dimers connected to multimodal imaging subunits. These multimodal agents comprise a fluorescent dye for OI and the chelator ((1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) (NODA-GA) for PET radiometal isotope labelling. Special focus was put on the influence of the used dyes on the properties of the whole bioconjugates. For this, several compounds with different fluorescent dyes and non-dye carrying subunits were synthesized and investigated. As fluorescent dyes, dansyl, NBD, derivatives of fluorescein, coumarin and rhodamine as well as three pyrilium-based dyes were employed. Considerable influence of the charge of the colored unit on hydrophilicity as well as in vitro target receptor binding was observed and classified. High radiochemical yields and purities were found during radiolabeling of the multimodal imaging subunits as well as their GRPR-specific bioconjugates with ⁶⁸Ga. Examinations of the photophysical properties of both molecule species displayed no loss or alteration of fluorescence characteristics.     

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.     

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.     

Fluorous Tagged Peptides for Intracellular Delivery and Biomedical Imaging

Fluorous tagged peptides have shown promising features for biomedical applications such as drug delivery and multimodal imaging. The bioconjugation of fluoroalkyl ligands onto cargo peptides greatly enhances their proteolytic stability and membrane penetration via a proposed “fluorine effect”. The tagged peptides also efficiently deliver other biomolecules such as DNA and siRNA into cells via a co-assembly strategy. The fluoroalkyl chains on peptides with antifouling properties enable efficient gene delivery in the presence of serum proteins. Besides intracellular biomolecule delivery, the amphiphilic peptides can be used to stabilized perfluorocarbon-filled microbubbles for ultrasound imaging. The fluorine nucleus on fluoroalkyls provides intrinsic probes for background-free magnetic resonance imaging. Labeling of fluorous tags with radionuclide ¹⁸F also allows tracing the biodistribution of peptides via positron emission tomography imaging. This mini-review will discuss properties and mechanism of the fluorous tagged peptides in these applications.     

Synthesis and Evaluation of [18F]‐Ammonium BODIPY Dyes as Potential Positron Emission Tomography Agents for Myocardial Perfusion Imaging

Recently, we demonstrated the potential of a [¹⁸F]‐trimethylammonium BODIPY dye for cardiac imaging. This is the first example of the use of the [¹⁸F]‐ammonium BODIPY dye for positron emission tomography (PET) myocardial perfusion imaging (MPI). In this report, we extend our study to other ammonium BODIPY dyes with different nitrogen substituents. These novel ammonium BODIPY dyes were successfully prepared and radiolabeled by the SnCl₄‐assisted ¹⁸F–¹⁹F isotopic exchange method. The microPET results and the biodistribution data reveal that nitrogen substituent changes have a significant effect on the in vivo and pharmacological properties of the tracers. Of the novel [¹⁸F]‐ammonium BODIPY dyes prepared in this work, the [¹⁸F]‐dimethylethylammonium BODIPY is superior in terms of myocardium uptake and PET imaging contrast. These results support our hypothesis that the ammonium BODIPY dyes have a great potential for use as PET/optical dual‐modality MPI probes.     

Comparison of [18F]F-CNBI and [18F]F-CNPIFE as Positron Emission Tomography Probes for Noninvasive Imaging of Glycogen Synthase Kinase-3 in Normal Mice

Glycogen synthase kinase-3 α/β is involved in dysregulation of neuronal tau protein in Alzheimer's disease (AD). There is an unmet clinical need for a blood-brain barrier (BBB) permeable positron emission tomography (PET) probe for imaging of GSK-3α/β in the brain to understand the pathogenesis of AD. Herein, we synthesized two PET probes, [¹⁸F]F-CNBI and [¹⁸F]F-CNPIFE, and evaluated their BBB permeability and affinity towards GSK-3α/β. [¹⁹F]F-CNPIFE showed higher in-vitro binding towards GSK-3α/β (IC₅₀=19.4±2.5 nM; n=3, for GSK-3α, IC₅₀=19.4±3.8 nM; n=3, for GSK-3β) compared to [¹⁹F]F-CNBI (IC₅₀=107.6±26.0 nM; n=4, for GSK-3α, IC₅₀=105.3±18.2 nM; n=3, for GSK-3β). [¹⁸F]F-CNPIFE showed 9.5-fold higher brain uptake than [¹⁸F]F-CNBI, in normal FVB/NJ mice, which was increased by additional 1.5-fold on co-administration of [¹⁹F]F-CNPIFE with respect to [¹⁸F]F-CNBI. Overall, [¹⁸F]F-CNPIFE is a promising PET probe for GSK-3α/β imaging and warrants further evaluation in an AD mouse model.     

Activatable 19F MRI Nanoparticle Probes for the Detection of Reducing Environments

¹⁹F magnetic resonance imaging (MRI) probes that can detect biological phenomena such as cell dynamics, ion concentrations, and enzymatic activity have attracted significant attention. Although perfluorocarbon (PFC) encapsulated nanoparticles are of interest in molecular imaging owing to their high sensitivity, activatable PFC nanoparticles have not been developed. In this study, we showed for the first time that the paramagnetic relaxation enhancement (PRE) effect can efficiently decrease the ¹⁹F NMR/MRI signals of PFCs in silica nanoparticles. On the basis of the PRE effect, we developed a reduction‐responsive PFC‐encapsulated nanoparticle probe, FLAME‐SS‐Gd³⁺ (FSG). This is the first example of an activatable PFC‐encapsulated nanoparticle that can be used for in vivo imaging. Calculations revealed that the ratio of fluorine atoms to Gd³⁺ complexes per nanoparticle was more than approximately 5.0×10², resulting in the high signal augmentation.     

Synthesis of gemini surfactants with twelve symmetric fluorine atoms and one singlet F MR signal as novel F MRI agents

A family of fluorinated gemini surfactants derived from perfluoropinacol has been synthesized as novel ¹⁹F magnetic resonance imaging (¹⁹F MRI) agents. These fluorinated surfactants with 12 symmetric fluorine atoms and one singlet ¹⁹F MR peak can be conveniently prepared from perfluoropinacol and oligo(ethylene glycols) on multi-gram scales. Solubility, hydrophilicity (log P), and critical micelle concentration (CMC) measurements of these fluorinated surfactants indicated that high aqueous solubility can be achieved by introducing oligo(ethylene glycols) with appropriate length into perfluoropinacol, i.e., manipulating the fluorine content (F%). One of these fluorinated surfactants with high aqueous solubility and excellent ¹⁹F MR properties has been identified by ¹⁹F MRI phantom experiments as a promising ¹⁹F MRI agent.     

Applications of “Hot” and “Cold” Bis(thiosemicarbazonato) Metal Complexes in Multimodal Imaging

The applications of coordination chemistry to molecular imaging has become a matter of intense research over the past 10 years. In particular, the applications of bis(thiosemicarbazonato) metal complexes in molecular imaging have mainly been focused on compounds with aliphatic backbones due to the in vivo imaging success of hypoxic tumors with PET (positron emission tomography) using ⁶⁴CuATSM [copper (diacetyl‐bis(N4‐methylthiosemicarbazone))]. This compound entered clinical trials in the US and the UK during the first decade of the 21^st century for imaging hypoxia in head and neck tumors. The replacement of the ligand backbone to aromatic groups, coupled with the exocyclic N's functionalization during the synthesis of bis(thiosemicarbazones) opens the possibility to use the corresponding metal complexes as multimodal imaging agents of use, both in vitro for optical detection, and in vivo when radiolabeled with several different metallic species. The greater kinetic stability of acenaphthenequinone bis(thiosemicarbazonato) metal complexes, with respect to that of the corresponding aliphatic ATSM complexes, allows the stabilization of a number of imaging probes, with special interest in “cold” and “hot” Cu(II) and Ga(III) derivatives for PET applications and ¹¹¹In(III) derivatives for SPECT (single‐photon emission computed tomography) applications, whilst Zn(II) derivatives display optical imaging properties in cells, with enhanced fluorescence emission and lifetime with respect to the free ligands. Preliminary studies have shown that gallium‐based acenaphthenequinone bis(thiosemicarbazonato) complexes are also hypoxia selective in vitro, thus increasing the interest in them as new generation imaging agents for in vitro and in vivo applications.     

Multimodal Functional Imaging for Cancer/Tumor Microenvironments Based on MRI,EPRI, and PET

Radiation therapy is one of the main modalities to treat cancer/tumor. The response to radiation therapy, however, can be influenced by physiological and/or pathological conditions in the target tissues, especially by the low partial oxygen pressure and altered redox status in cancer/tumor tissues. Visualizing such cancer/tumor patho-physiological microenvironment would be a useful not only for planning radiotherapy but also to detect cancer/tumor in an earlier stage. Tumor hypoxia could be sensed by positron emission tomography (PET), electron paramagnetic resonance (EPR) oxygen mapping, and in vivo dynamic nuclear polarization (DNP) MRI. Tissue oxygenation could be visualized on a real-time basis by blood oxygen level dependent (BOLD) and/or tissue oxygen level dependent (TOLD) MRI signal. EPR imaging (EPRI) and/or T₁-weighted MRI techniques can visualize tissue redox status non-invasively based on paramagnetic and diamagnetic conversions of nitroxyl radical contrast agent. ¹³C-DNP MRI can visualize glycometabolism of tumor/cancer tissues. Accurate co-registration of those multimodal images could make mechanisms of drug and/or relation of resulted biological effects clear. A multimodal instrument, such as PET-MRI, may have another possibility to link multiple functions. Functional imaging techniques individually developed to date have been converged on the concept of theranostics.     

Aryl‐Phosphonate Lanthanide Complexes and Their Fluorinated Derivatives: Investigation of Their Unusual Relaxometric Behavior and Potential Application as Dual Frequency 1H/19F MRI Probes

A series of low molecular weight lanthanide complexes were developed that have high ¹H longitudinal relaxivities (r₁) and the potential to be used as dual frequency ¹H and ¹⁹F MR probes. Their behavior was investigated in more detail through relaxometry, pH‐potentiometry, luminescence, and multinuclear NMR spectroscopy. Fitting of the ¹H NMRD and ¹⁷O NMR profiles demonstrated a very short water residence lifetime (<10 ns) and an appreciable second sphere effect. At lower field strengths (20 MHz), two of the complexes displayed a peak in r₁ (21.7 and 16.3 mM ^?1 s^?1) caused by an agglomeration, that can be disrupted through the addition of phosphate anions. NMR spectroscopy revealed that at least two species are present in solution interconverting through an intramolecular binding process. Two complexes provided a suitable signal in ¹⁹F NMR spectroscopy and through the selection of optimized imaging parameters, phantom images were obtained in a MRI scanner at concentrations as low as 1 mM . The developed probes could be visualized through both ¹H and ¹⁹F MRI, showing their capability to function as dual frequency MRI contrast agents.     

Introduction to Infrared and Raman-Based Biomedical Molecular Imaging and Comparison with Other Modalities

Molecular imaging has rapidly developed to answer the need of image contrast in medical diagnostic imaging to go beyond morphological information to include functional differences in imaged tissues at the cellular and molecular levels. Vibrational (infrared (IR) and Raman) imaging has rapidly emerged among the molecular imaging modalities available, due to its label-free combination of high spatial resolution with chemical specificity. This article presents the physical basis of vibrational spectroscopy and imaging, followed by illustration of their preclinical in vitro applications in body fluids and cells, ex vivo tissues and in vivo small animals and ending with a brief discussion of their clinical translation. After comparing the advantages and disadvantages of IR/Raman imaging with the other main modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography/single-photon emission-computed tomography (PET/SPECT), ultrasound (US) and photoacoustic imaging (PAI), the design of multimodal probes combining vibrational imaging with other modalities is discussed, illustrated by some preclinical proof-of-concept examples.     

Partially Fluorinated Copolymers as Oxygen Sensitive 19F MRI Agents

Effective diagnosis of disease and its progression can be aided by ¹⁹F magnetic resonance imaging (MRI) techniques. Specifically, the inherent sensitivity of the spin–lattice relaxation time (T₁) of ¹⁹F nuclei to oxygen partial pressure makes ¹⁹F MRI an attractive non-invasive approach to quantify tissue oxygenation in a spatiotemporal manner. However, there are only few materials with the adequate sensitivity to be used as oxygen-sensitive ¹⁹F MRI agents at clinically relevant field strengths. Motivated by the limitations in current technologies, we report highly fluorinated monomers that provide a platform approach to realize water-soluble, partially fluorinated copolymers as ¹⁹F MRI agents with the required sensitivity to quantify solution oxygenation at clinically relevant magnetic field strengths. The synthesis of a systematic library of partially fluorinated copolymers enabled a comprehensive evaluation of copolymer structure–property relationships relevant to ¹⁹F MRI. The highest-performing material composition demonstrated a signal-to-noise ratio that corresponded to an apparent ¹⁹F density of 220 mm , which surpasses the threshold of 126 mm ¹⁹F required for visualization on a three Tesla clinical MRI. Furthermore, the T₁ of these high performing materials demonstrated a linear relationship with solution oxygenation, with oxygen sensitivity reaching 240×10⁻⁵ mmHg⁻¹s⁻¹. The relationships between material composition and ¹⁹F MRI performance identified herein suggest general structure–property criteria for the further improvement of modular, water-soluble ¹⁹F MRI agents for quantifying oxygenation in environments relevant to medical imaging.     

Towards Enhanced MRI Performance of Tumor-Specific Dimeric Phenylboronic Contrast Agents

It is known that phenylboronic acid (PBA) can target tumor tissues by binding to sialic acid, a substrate overexpressed by cancer cells. This capability has previously been explored in the design of targeting diagnostic probes such as Gd- and ⁶⁸Ga-DOTA-EN-PBA, two contrast agents for magnetic resonance imaging (MRI) and positron emission tomography (PET), respectively, whose potential has already been demonstrated through in vivo experiments. In addition to its high resolution, the intrinsic low sensitivity of MRI stimulates the search for more effective contrast agents, which, in the case of small-molecular probes, basically narrows down to either increased tumbling time of the entire molecule or elevated local concentration of the paramagnetic ions, both strategies resulting in enhanced relaxivity, and consequently, a higher MRI contrast. The latter strategy can be achieved by the design of multimeric Gd^III complexes. Based on the monomeric PBA-containing probes described recently, herein, we report the synthesis and characterization of the dimeric analogues (Gd^III-DOTA-EN)₂-PBA and (Gd^III-DOTA-EN)₂F₂PBA. The presence of two Gd ions in one molecule clearly contributes to the improved biological performance, as demonstrated by the relaxometric study and cell-binding investigations.     

Emerging applications of near-infrared fluorescent metal nanoclusters for biological imaging

Recent advances in the development of near-infrared fluorescent metal nanoclusters for bioimaging applications have been thoroughly overviewed.     

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.     

Cyclotriphosphazene,a scaffold for 19F MRI contrast agents

A cyclotriphosphazene substituted with six 3,5-bis(trifluoromethyl) benzyloxy units was designed as a novel ¹⁹F MRI contrast agent. The resulting molecule has 36 magnetically equivalent fluorine atoms and exhibited suitable MRI properties with high imaging sensitivity, confirming the proof-of-concept as a convenient scaffold for the production of new ¹⁹F MRI contrasts agents.     

<Superscript>68</Superscript>Ga-radiolabeled magnetic nanoparticles for PET–MRI imaging

In this study, magnetic multimodal nanoparticles with potential applications in magnetic- and nuclear-medicine imaging, magnetic resonance imaging, hyperthermia, and theranostic (therapeutic and diagnostic), applications were prepared by coating iron oxide nanoparticles with silica (core–shell), functionalizing with aminopropyltriethoxy silane and coupling with diethylenetriamine pentaacetic acid ligand (DTPA). Radiolabeling of core–shell–DTPA particles with ⁶⁸Ga radiometal was carried out through chelation of ⁶⁸Ga(III) ions by DTPA and was used for positron emission tomography. The biodistribution of the ⁶⁸Ga-radiolabeled magnetic nanoparticles compared to free ⁶⁸Ga(III) was checked in normal Balb/c mice up to 2 h.