Organic Nanoprobes for Fluorescence and 19F Magnetic Resonance Dual‐Modality Imaging

Multimodal imaging techniques have been demonstrated to be greatly advantageous in achieving accurate diagnosis and gained increasing attention in recent decades. Herein, we present a new strategy to integrate the complementary modalities of ¹⁹F magnetic resonance imaging (¹⁹F MRI) and fluorescence imaging (FI) into a polymer nanoprobe composed of hydrophobic fluorescent organic core and hydrophilic fluorinated polymer shell. The alkyne‐terminated fluorinated copolymer (Pn) of 2,2,2‐trifluoroethyl acrylate (TFEA) and poly(ethylene glycol) methyl ether acrylate (PEGA) was first prepared via atom transfer radical polymerization (ATRP). The PEGA plays an important role in both improving ¹⁹F signal and modulating the hydrophilicity of Pn. The alkynyl tail in Pn is readily conjugated with azide modified tetra‐phenylethylene (TPE) through click chemistry to form azo polymer (TPE‐azo‐Pn). The core‐shell nanoprobes (TPE‐P3N) with an average particle size of 57.2 ± 8.8 nm are obtained via self‐assembly with ultrasonication in aqueous solution. These nanoprobes demonstrate high water stability, good biocompatibility, strong fluorescence and good ¹⁹F MRI performance, which present great potentials for simultaneous fluorescence imaging and ¹⁹F–MR imaging.     

Multifunctional Nanoprobe for MRI/Optical Dual‐Modality Imaging and Radical Scavenging

The development of novel nanomaterials for the diagnosis and/or treatment of human diseases has become an important issue. In this work, a multifunctional theranostic agent was designed by covalently binding hydroxyl‐ and amino‐bearing C₆₀ derivatives (C₆₀O_～10(OH)_～16(NH₂)_～6(NO₂)_～6 ? 24 H₂O) with gadolinium diethylenetriaminepentaacetic acid (Gd‐DTPA) to yield C₆₀O_～10(OH)_～16(NH₂)_～6(NO₂)_～6 ? 24 H₂O/(Gd‐DTPA)₃ ( DF₁Gd₃ ). The obtained DF₁Gd₃ shows more than fourfold contrast improvement over commercial Gd‐DTPA along with multiwavelength fluorescent emission for dual‐modality diagnosis. An inner‐ear magnetic resonance imaging (MRI) study was designed as a model of biological barriers, including the blood/brain barrier (BBB) for DF₁Gd₃ to investigate its in vivo behavior. This revealed that the fabricated contrast agent dramatically increases the local contrast but can not cross the middle ear/inner ear barrier and endolymph/perilymph barrier in the inner ear, and thus it is also BBB‐prohibited in normal individuals. In vivo biodistribution studies suggested that 1) DF₁Gd₃ could circulate in vessels for a relatively long time and is mainly eliminated through liver and kidney, 2) DF₁Gd₃ may potentially function as a liver‐specific MRI contrast agent. Interestingly, DF₁Gd₃ also shows an excellent quenching effect on hydroxyl radicals, as revealed by the DMPO spin trap/ESR method. The combination of enhanced MRI/FL imaging and local treatment of lesions is unique to DF₁Gd₃ and potentiates the medical paradigm of “detect and treat/prevent” in combating human diseases related to reactive oxygen.     

Dual‐Frequency Calcium‐Responsive MRI Agents

Responsive or smart magnetic resonance imaging (MRI) contrast agents are molecular sensors that alter the MRI signal upon changes in a particular parameter in their microenvironment. Consequently, they could be exploited for visualization of various biochemical events that take place at molecular and cellular levels. In this study, a set of dual‐frequency calcium‐responsive MRI agents are reported. These are paramagnetic, fluorine‐containing complexes that produce remarkably high MRI signal changes at the ¹H and ¹⁹F frequencies at varying Ca²⁺ concentrations. The nature of the processes triggered by Ca²⁺ was revealed, allowing a better understanding of these complex systems and their further improvement. The findings indicate that these double‐frequency tracers hold great promise for development of novel functional MRI methods.     

Semiconducting Polymer Dots with Dual‐Enhanced NIR‐IIa Fluorescence for Through‐Skull Mouse‐Brain Imaging

Fluorescence probes in the NIR‐IIa region show drastically improved imaging owing to the reduced photon scattering and autofluorescence in biological tissues. Now, NIR‐IIa polymer dots (Pdots) are developed with a dual fluorescence enhancement mechanism. First, the aggregation induced emission of phenothiazine was used to reduce the nonradiative decay pathways of the polymers in condensed states. Second, fluorescence quenching was minimized by different levels of steric hindrance to further boost the fluorescence. The resulting Pdots displayed a fluorescence QY of ca. 1.7 % in aqueous solution, suggesting an enhancement of ca. 21 times in comparison with the original polymer in tetrahydrofuran (THF) solution. Small‐animal imaging by using the NIR‐IIa Pdots exhibited a remarkable improvement in penetration depth and signal to background ratio, as confirmed by through‐skull and through‐scalp fluorescent imaging of the cerebral vasculature of live mice.     

All‐in‐One Target‐Cell‐Specific Magnetic Nanoparticles for Simultaneous Molecular Imaging and siRNA Delivery

Cancer‐cell‐targeted gene silencing was observed with a magnetic‐nanoparticle platform (MEIO, magnetism‐engineered iron oxide) on which a fluorescent dye, siRNA, and a RGD‐peptide targeting moiety were attached (see picture). The different functionalities enable the macroscopic (magnetic resonance) and microscopic (fluorescence) imaging of target cells. This system may be suitable for concurrent diagnostic and therapeutic applications.

     

Dual‐Phase Thermally Activated Delayed Fluorescence Luminogens: A Material for Time‐Resolved Imaging Independent of Probe Pretreatment and Probe Concentration

Developing luminescent probes with long lifetime and high emission efficiency is essential for time‐resolved imaging. However, the practical applications usually suffer from emission quenching of traditional luminogens in aggregated states, or from weak emission of aggregation‐induced emission type luminogens in monomeric states. Herein, we overcome this dilemma by a rigid‐and‐flexible alternation design in donor–acceptor–donor skeletons, to achieve a thermally activated delayed fluorescence luminogen with high emission efficiency both in the monomeric state (quantum yield up to 35.3 %) and in the aggregated state (quantum yield up to 30.8 %). Such a dual‐phase strong and long‐lived emission allows a time‐resolved luminescence imaging, with an efficiency independent of probe pretreatment and probe concentration. The findings open opportunities for developing luminescent probes with a usage in larger temporal and spatial scales.     

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.     

Real‐Time Fluorescence Turn‐On Detection of Alkaline Phosphatase Activity with a Novel Perylene Probe

A tetracationic perylene probe (probe 2 ) was designed and synthesized. Probe 1 was used for the real‐time fluorescence turn‐on assay of alkaline phosphatase (ALP) activity and inhibitor screening. Probe 1 monomer fluorescence could be very efficiently quenched by ATP through the formation of an ATP/probe 1 complex. ALP triggered the degradation of ATP, the breakdown of the ATP/probe 1 complex, and the recovery of the probe 1 monomer fluorescence. In the presence of an ALP inhibitor, a decrease in fluorescence recovery was observed.     

磁共振/光学双模态分子探针的研究现状与展望

基于磁共振与荧光成像的双模态成像技术不仅克服了传统单一分子影像技术在灵敏度、特异度、分辨率等方面的固有缺陷，更是拓宽了分子影像技术在诊断及治疗监控等领域的研究范围及应用前景。本文将对磁共振/荧光双模态分子探针的应用情况和研究进展等进行综述。     

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.     

一种用于细胞核成像的新型双光子荧光探针

本文报道了具有大双光子活性吸收截面的DNA探针BMVEC, 同时首次利用双光子显微镜完成了与DAPI的复染实验, 实验结果证实了BMVEC对细胞核的选择性标记能力.     

发夹型荧光分子探针用于DNA甲基化酶的活性分析及其在药物筛选中的应用

报道了一种基于发夹型荧光探针的甲基化酶活性的分析方法, 甲基化酶和相应的限制性内切酶的识别位点被设计在发夹型探针的茎部, 四甲基罗丹明(TAMRA)被连接在探针的5'端, 其荧光被连在3'端的熄灭基团4-(4'-二甲基对胺基偶氮苯)苯甲酸(DABCYL)所熄灭. 限制性内切酶可切割未发生甲基化修饰的探针, 导致探针的发夹结构遭到破坏, 引起TAMRA荧光信号的恢复. 根据荧光信号的恢复程度可实现对甲基化酶活性的分析. 在此基础上, 建立了一种简便、快速分析抗肿瘤药物对DNA甲基化酶活性的影响的方法, 为筛选针对基因甲基化异常引起的恶性肿瘤的治疗药物提供了一种新的思路和方法.     

19F MRI Probes for Multimodal Imaging**

Magnetic resonance imaging (MRI) is one of the most powerful imaging tools today, capable of displaying superior soft-tissue contrast. This review discusses developments in the field of ¹⁹F MRI multimodal probes in combination with optical fluorescence imaging (OFI), ¹H MRI, chemical exchange saturation transfer (CEST) MRI, ultrasonography (USG), X-ray computed tomography (CT), single photon emission tomography (SPECT), positron emission tomography (PET), and photoacoustic imaging (PAI). In each case, multimodal ¹⁹F MRI probes compensate for the deficiency of individual techniques and offer improved sensitivity or accuracy of detection over unimodal counterparts. Strategies for designing ¹⁹F MRI multimodal probes are described with respect to their structure, physicochemical properties, biocompatibility, and the quality of images.     

Cyclodextrin‐Based Bimodal Fluorescence/MRI Contrast Agents: An Efficient Approach to Cellular Imaging

A novel bimodal fluorescence/MRI probe based on a cyclodextrin scaffold has been synthesized and characterized. The final agent employs the fluorescein (F) functionality as a fluorescence marker and the Gd^III complex of a macrocyclic DOTA‐based ligand (GdL) having one aminobenzyl‐phosphinic acid pendant arm as an MRI probe, and has a statistical composition of (GdL)_6.9‐F_0.1‐β‐CD. Slow rotational dynamics (governed by a very rigid cyclodextrin scaffold) combined with fast water exchange (ensured by the chosen macrocyclic ligand) resulted in a high relaxivity of ～22 s^?1 mM ^?1 per Gd^III or ～150 s^?1 mM ^?1 per molecule of the final conjugate (20 MHz, 25 °C). In vitro labelling of pancreatic islets (PIs) and rat mesenchymal stem cells has been successfully performed. The agent is not cytotoxic and is easily internalized into cells. The labelled cells can be visualized by MRI, as proved by the detection of individual labelled PIs. A fluorescence study performed on mesenchymal stem cells showed that the agent stays in the intracellular space for a long time.     

Highly Water‐Dispersible Surface‐Modified Gd2O3 Nanoparticles for Potential Dual‐Modal Bioimaging

Water‐dispersible and luminescent gadolinium oxide (GO) nanoparticles (NPs) were designed and synthesized for potential dual‐modal biological imaging. They were obtained by capping gadolinium oxide nanoparticles with a fluorescent glycol‐based conjugated carboxylate (H L ). The obtained nanoparticles (GO‐ L ) show long‐term colloidal stability and intense blue fluorescence. In addition, L can sensitize the luminescence of europium(III) through the so‐called antenna effect. Thus, to extend the spectral ranges of emission, europium was introduced into L‐ modified gadolinium oxide nanoparticles. The obtained Eu^III‐doped particles (Eu:GO‐ L ) can provide visible red emission, which is more intensive than that without L capping. The average diameter of the monodisperse modified oxide cores is about 4 nm. The average hydrodynamic diameter of the L ‐modified nanoparticles was estimated to be about 13 nm. The nanoparticles show effective longitudinal water proton relaxivity. The relaxivity values obtained for GO‐ L and Eu:GO‐ L were r₁=6.4 and 6.3 s^?1 mM ^?1 with r₂/r₁ ratios close to unity at 1.4 T. Longitudinal proton relaxivities of these nanoparticles are higher than those of positive contrast agents based on gadolinium complexes such as Gd‐DOTA, which are commonly used for clinical magnetic resonance imaging. Moreover, these particles are suitable for cellular imaging and show good biocompatibility.     

荧光/MRI双模态靶向成像诊疗试剂的制备

在聚乙二醇二胺(NH_2-PEG-NH_2)修饰的石墨烯量子点(GODs)表面以酰胺键偶联二乙基三胺五乙酸(DTPA)分子,之后将Gd~(3+)离子与其进行配合,得到了GODs-Gd(DTPA)复合纳米粒子,然后再通过酰胺键在GODs-Gd(DTPA)的表面修饰叶酸(FA)靶分子,最后进一步将阿霉素(DOX)通过π-π堆垛吸附在造影剂的表面,制备了FA/GODs-Gd(DTPA)/DOX荧光/MRI双模态靶向肺癌细胞成像诊疗试剂,通过透射电子显微镜、紫外可见吸收光谱、荧光光谱和激光共聚焦扫描显微镜等手段表征了其形貌、发光性能和靶向成像性能。MRI、激光共聚焦扫描显微镜和MTT等结果表明,相对于正常的HLF细胞,所制备的FA/GODs-Gd(DTPA)/DOX纳米粒子能够靶向检测FA受体高表达的肺癌H460细胞,并具有明显的抗肿瘤活性。     

Correcting for Spectral Cross‐Talk in Dual‐Color Fluorescence Cross‐Correlation Spectroscopy

Dual‐color fluorescence cross‐correlation spectroscopy (dcFCCS) allows one to quantitatively assess the interactions of mobile molecules labeled with distinct fluorophores. The technique is widely applied to both reconstituted and live‐cell biological systems. A major drawback of dcFCCS is the risk of an artifactual false‐positive or overestimated cross‐correlation amplitude arising from spectral cross‐talk. Cross‐talk can be reduced or prevented by fast alternating excitation, but the technology is not easily implemented in standard commercial setups. An experimental strategy is devised that does not require specialized hardware and software for recognizing and correcting for cross‐talk in standard dcFCCS. The dependence of the cross‐talk on particle concentrations and brightnesses is quantitatively confirmed. Moreover, it is straightforward to quantitatively correct for cross‐talk using quickly accessible parameters, that is, the measured (apparent) fluorescence count rates and correlation amplitudes. Only the bleed‐through ratio needs to be determined in a calibration measurement. Finally, the limitations of cross‐talk correction and its influence on experimental error are explored.