Fluorescent,Recombinant-Protein-Conjugated,Near-Infrared-Emitting Quantum Dots for in Vitro and in Vivo Dual-Color Molecular Imaging

Near-infrared (NIR)-emitting fluorescent probes are widely used for molecular imaging at the whole-body level. However, NIR-emitting fluorescent probes emitting over λ=700 nm are not suitable for molecular imaging at the cellular level, because most of the conventional fluorescence microscopes have very low optical sensitivity in the NIR region. Thus, to achieve fluorescence imaging at the cellular and whole-body levels by using single probes, visible and NIR-emitting dual-color fluorescent probes are desirable. For dual-color fluorescence molecular imaging, we synthesized fluorescent, recombinant-protein-conjugated, NIR-emitting quantum dots (QDs), in which the recombinant protein consists of enhanced green fluorescent protein (EGFP) and the immunoglobulin binding domain (B1) of protein G. This dual-color fluorescent QD probe binds the Fc region of immunoglobulin G (IgG) through its B1 domain at the QD surface and acts as a molecular-imaging probe at both the cellular and whole-body levels. In this paper, we present the synthesis of fluorescent, recombinant protein (HisEGFP-GB1)-conjugated, NIR-emitting QDs and their application to the dual-color molecular imaging of breast cancer cells in vitro and in vivo.     

In Vivo Evaluation of Sgc8-c Aptamer as a Molecular Imaging Probe for Colon Cancer in a Mouse Xenograft Model

Recent biotechnological applications in the field of clinical oncology led to the identification of new biomarkers as molecular targets of cancer, and to broad developments in the field of personalized medicine. Aptamers are oligonucleotides (ssDNA or RNA) that are selected to specifically recognize a molecular target with high affinity and specificity. Based on this, new horizons for their use as molecular imaging probes are being explored. The objective of this work was to evaluate the Sgc8-c aptamer conjugated with Alexa Fluor 647 fluorophore as an imaging probe in a colon tumor xenograft mouse model, with potential application in molecular imaging. In this study, the LS174T cell line was used to induce colorectal adenocarcinoma in nude mice. After confirmation of PTK7 overexpression by immunohistochemistry, in vivo studies were performed. Pharmacokinetic, in vivo and ex vivo biodistribution imaging, and a competition assay were evaluated by fluorescence imaging. In vivo visualization of the probe in the tumors was assessed two hours after aptamer probe administration, exhibiting excellent tumor-to-background ratios in biodistribution studies and high specificity in the competition test. Our results demonstrated the functionality of Scg8-c as an imaging probe for colon cancer, with potential clinical applications.     

New Developments in Carbonic Anhydrase IX-Targeted Fluorescence and Nuclear Imaging Agents

Carbonic anhydrase IX (CAIX) is a tumor-specific and hypoxia-induced biomarker for the molecular imaging of solid malignancies. The nuclear- and optical-imaging of CAIX-expressing tumors have received great attention due to their potential for clinical applications. Nuclear imaging is a powerful tool for the non-invasive diagnosis of primary and metastatic CAIX-positive tumors and for the assessment of responses to antineoplastic treatment. Intraoperative optical fluorescence imaging provides improved visualization for surgeons to increase the discrimination of tumor lesions, allowing for safer surgical treatment. Over the past decades, many CAIX-targeted molecular imaging probes, based on monoclonal antibodies, antibody fragments, peptides, and small molecules, have been reported. In this review, we outline the recent development of CAIX-targeted probes for single-photon emission computerized tomography (SPECT), positron emission tomography (PET), and near-infrared fluorescence imaging (NIRF), and we discuss issues yet to be addressed.     

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell-penetrating peptides (CPPs) and pore-forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)- with DNA-based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP–FIT PNA conjugates were affected. Live-cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob-like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin-O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA-based FIT probes were brighter and more responsive than PNA-based FIT probes. Optimized conditions enabled live-cell multicolor imaging of three different mRNA target sequences.     

Chloro‐Functionalized Photo‐crosslinking BODIPY for Glutathione Sensing and Subcellular Trafficking

Glutathione (GSH) is one of major antioxidants inside cells that regulates oxidoreduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensors and do not provide details of cellular localization. A new fluorescent probe (pcBD2‐Cl), which is cell permeable and selectively reacts with GSH in situ, has been developed. The in situ GSH‐labeled probe (pcBD2–GSH) exhibited quenches fluorescence, but subsequent binding to cellular abundant glutathione S‐transferase (GST) recovers the fluorescence intensity, which makes it possible to image the GSH–GST complex in live cells. Interactions between probe and GST were confirmed by means of photo‐crosslinking under intact live‐cell conditions. Interestingly, isomers of chloro‐functionalized 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) compounds behaved very distinctively inside the cells. Following co‐staining imaging with MitoTracker and mitochondria fractionation upon lipopolysaccharide‐mediated reactive oxygen species induction experiments showed that pcBD2–GSH accumulated in mitochondria. This is the first example of a live‐cell imaging probe to visualize translocation of GSH from the cytosol to mitochondria.     

Highly specific targeting and imaging of live cancer cells by using a peptide probe developed from rationally designed peptides

Specific detection and in vivo tracing of cancer biomarkers are important for cancer analysis. In this work, a simple and effective strategy for developing peptide probes was established. Peptides were rationally designed by using an antisense peptide approach directed towards an extracellular fragment (EL2) of a novel tumor-related protein LAPTM4B. Positional-scanning and stepwise affinity screening was employed to obtain an optimal peptide AP2H (IHGHHIISVG). The dissociation constant between the two small peptides, AP2H and the target EL2, was 5.51 μM under physiological conditions. Fluorescence imaging assays indicated that AP2H can recognize live hepatoma cells by targeting the LAPTM4B protein on the cell surface with high specificity, low cytotoxicity and desirable cell penetrability. Compared to negative control cells, AP2H could differentiate cells with different expression levels of LAPTM4B. The screened peptide probe for molecular signatures of cancer cells, based on targeting the LAPTM4B protein, has potential applications in cancer diagnosis and targetable drug delivery.     

A Cyanine-Bridged Somatostatin Hybrid Probe for Multimodal SSTR2 Imaging in Vitro and in Vivo: Synthesis and Evaluation

Multimodal imaging probes have attracted the interest of ongoing research, for example, for the surgical removal of tumors. Modular synthesis approaches allow the construction of hybrid probes consisting of a radiotracer, a fluorophore and a targeting unit. We present the synthesis of a new asymmetric bifunctional cyanine dye that can be used as a structural and functional linker for the construction of such hybrid probes. ⁶⁸Ga-DOTATATE, a well-characterized radiopeptide targeting the overexpressed somatostatin receptor subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our cyanine dye, thus providing additional information along with the data obtained from the radiotracer. We tested the SSTR2-targeting and imaging properties of the resulting probe ⁶⁸Ga-DOTA-ICC-TATE in vitro and in a tumor xenograft mouse model. Despite the close proximity between dye and pharmacophore, we observed a high binding affinity towards SSTR2 as well as elevated uptake in SSTR2-overexpressing tumors in the positron emission tomography (PET) scan and histological examination.     

Single Labeled DNA FIT Probes for Avoiding False‐Positive Signaling in the Detection of DNA/RNA in qPCR or Cell Media

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real‐time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance‐dependent interaction between a fluorophore and another label. Such duallabeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label–label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, “DNA FIT probes”, that are designed to avoid false‐positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the “TO base” in cis with the disordered nucleobases of the single strand, and 2) intercalation of the “TO nucleotide” with double strands in trans. However, formation of the probe–target duplex enforces stacking and increases the fluorescence of the TO base. We explored open‐chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe–target complexes. DNA and RNA targets provided up to 12‐fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false‐positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.     

Imaging target mRNA and siRNA-mediated gene silencing in vivo with ribozyme-based reporters

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.     

甘氨酸-氟硼荧近红外荧光分子探针的合成及性能测试

将一种具有近红外荧光性能的氟硼荧染料（BDDIPY）作为显影基团,制成新型的检测肿瘤细胞的近红外荧光分子探针。首先以氟硼二吡咯烷（BOD）为起始原料,通过缩合反应,将具有强供电子作用的N-苯基亚氨基二苄引入到氟硼荧中,得到了荧光光谱处于近红外区的氟硼荧化合物,接着用N-羟基琥珀酰亚胺为活化剂,将具有肿瘤亲和力的甘氨酸引入到荧光基团-氟硼荧（BDDIPY）,最终得到甘氨酸-氟硼荧近红外荧光探针,并对其结构进行了核磁和质谱表征,并进行了光学和生物学性能测试。实验结果显示：甘氨酸-氟硼荧近红外荧光分子探针的荧光发射波长大于700 nm,与肺癌细胞（GLC-82）具有较好的亲和力,达到23.65,是一个具有良好应用前景的检测肿瘤细胞的近红外荧光分子探针。     

反应激活型酶荧光探针的研究进展

酶在维持生物体内稳态与生命活动的正常运行方面发挥着举足轻重的作用。某些特定酶含量及活性的异常与人类重大疾病的发生与发展密切相关。因此,生物体内特定酶的实时原位检测及可视化成像具有重要的意义。化学荧光探针具有选择性好、灵敏度高及高时空分辨率可视化成像等优点,近年来研究者设计合成了大量的可用于生物体系内酶识别与可视化成像的荧光探针。目前识别酶的荧光探针主要有两类：（1）基于酶对荧光探针分子中酶抑制剂基团的识别引起探针荧光信号的变化;（2）基于酶对荧光探针特异性催化反应来实现识别前后荧光信号的激活,称为反应激活型酶荧光探针。对反应激活型酶荧光探针的设计策略及4种重大疾病相关的生物标志酶（单胺氧化酶、β-半乳糖苷酶、硝基还原酶、γ-谷氨酰转肽酶）的识别可视化荧光探针研究进展进行了综述,对未来酶识别荧光探针的研究方向进行了展望。     

Intravascular targets for molecular contrast-enhanced ultrasound imaging

Molecular targeting of contrast agents for ultrasound imaging is emerging as a new medical imaging modality. It combines advances in ultrasound technology with principles of molecular imaging, thereby allowing non-invasive assessment of biological processes in vivo. Preclinical studies have shown that microbubbles, which provide contrast during ultrasound imaging, can be targeted to specific molecular markers. These microbubbles accumulate in tissue with target (over) expression, thereby significantly increasing the ultrasound signal. This concept offers safe and low-cost imaging with high spatial resolution and sensitivity. It is therefore considered to have great potential in cancer imaging, and early-phase clinical trials are ongoing. In this review, we summarize the current literature on targets that have been successfully imaged in preclinical models using molecularly targeted ultrasound contrast agents. Based on preclinical experience, we discuss the potential clinical utility of targeted microbubbles.     

Exploring BODIPY-Based Sensor for Imaging of Intracellular Microviscosity in Human Breast Cancer Cells

BODIPY-based molecular rotors are highly attractive imaging tools for imaging intracellular microviscosity in living cells. In our study, we investigated the ability to detect the microviscosity of biological objects by using BDP-NO₂ and BDP-H molecular rotors. We describe in detail the optical properties of BDP-NO₂ and BDP-H molecular rotors in aqueous media with and without proteins, together with their accumulation dynamics and localization in live and fixed human breast cancer cells. Furthermore, we investigate the applicability of these molecules to monitor microviscosity in the organelles of human breast cancer cells by fluorescence lifetime imaging microscopy (FLIM). We demonstrate that the BDP-NO₂ molecular rotor aggregates in aqueous media and is incompatible with live cell imaging. The opposite effect is observed with BDP-H which preserves its stability in aqueous media, diffuses through the plasma membrane and accumulates in lipid droplets (LDs) and the cytosol of both live and fixed MCF-7 and MDA-MB-231 cancer cells. Finally, by utilizing BDP-H we demonstrate that LD microviscosity is significantly elevated in more malignant MDA-MB-231 human breast cancer cells, as compared to MCF-7 breast cancer cells. Our findings demonstrate that BDP-H is a water-compatible probe that can be successfully applied to measure microviscosity in the LDs of living cells.     

Pathophysiology and Molecular Imaging of Diabetic Foot Infections

Diabetic foot infection is the leading cause of non-traumatic lower limb amputations worldwide. In addition, diabetes mellitus and sequela of the disease are increasing in prevalence. In 2017, 9.4% of Americans were diagnosed with diabetes mellitus (DM). The growing pervasiveness and financial implications of diabetic foot infection (DFI) indicate an acute need for improved clinical assessment and treatment. Complex pathophysiology and suboptimal specificity of current non-invasive imaging modalities have made diagnosis and treatment response challenging. Current anatomical and molecular clinical imaging strategies have mainly targeted the host’s immune responses rather than the unique metabolism of the invading microorganism. Advances in imaging have the potential to reduce the impact of these problems and improve the assessment of DFI, particularly in distinguishing infection of soft tissue alone from osteomyelitis (OM). This review presents a summary of the known pathophysiology of DFI, the molecular basis of current and emerging diagnostic imaging techniques, and the mechanistic links of these imaging techniques to the pathophysiology of diabetic foot infections.     

比率型过氧化氢荧光探针的合成及成像应用

采用6-甲氧基苯并噻唑-2-羟基喹啉作为双光子荧光团、硼酸酯作为过氧化氢（H2O2）识别基团，合成比率型检测H2O2的双光子荧光探针{6-甲氧基-2-[6-(4,4,5,5-四甲基-1,3,2-二氧硼杂环戊烷-2-基)喹啉-2-基]苯并[d]噻唑}（MQH2O2）。利用荧光光谱和双光子荧光光谱对探针进行H2O2响应能力的评估，结果显示探针具有良好的H2O2比率响应（30 min内比率信号增强约25.4倍、检出限低至38.6 nmol/L）和双光子性质（最大双光子荧光活性截面为150 GM）。通过双光子共聚焦成像完成了细胞和大脑组织成像，结果表明该探针能够实现脑卒中诱导细胞氧化应激的原位成像分析。     

Detection and Imaging of Active Substances in Early Atherosclerotic Lesions Using Fluorescent Probes

Atherosclerosis (AS) is a vascular disease caused by chronic inflammation and lipids that is the main cause of myocardial infarction, stroke and other cardiovascular diseases. Atherosclerosis is often difficult to detect in its early stages due to the absence of clinically significant vascular stenosis. This is not conducive to early intervention or treatment of the disease. Over the past decade, researchers have developed various imaging methods for the detection and imaging of atherosclerosis. At the same time, more and more biomarkers are being found that can be used as targets for detecting atherosclerosis. Therefore, the development of a variety of imaging methods and a variety of targeted imaging probes is an important project to achieve early assessment and treatment of atherosclerosis. This paper provides a comprehensive review of the optical probes used to detect and target atherosclerosis imaging in recent years, and describes the current challenges and future development directions.     

Recent Advances in the Development of Optical Imaging Probes for γ-Glutamyltranspeptidase

γ-Glutamyltranspeptidase (GGT) is a cell-membrane-bound protease that participates in cellular glutathione and cysteine homeostasis, which are closely related to many physiological and pathological processes. The accurate measurement of GGT activity is useful for the early diagnosis of diseases. In the past few years, many efforts have been made to build optical imaging probes for the detection of GGT activity both in vitro and in vivo. In this Minireview, recent advances in the development of various optical imaging probes for GGT, including activatable fluorescence probes, ratiometric fluorescence probes, and activatable bioluminescence probes, are summarized. This review starts from the instruction of the GGT enzyme and its biological functions, followed by a discussion of activatable fluorescence probes that show off–on fluorescence in response to GGT. GGT-activatable two-photon fluorescence imaging probes with improved imaging depth and spatial resolution are also discussed. Ratiometric fluorescence probes capable of accurately reporting on GGT levels through a self-calibration mechanism are discussed, followed by describing GGT-activatable bioluminescence probes that can offer a high signal-to-background ratio to detect GGT in living mice. Finally, current challenges and further perspectives for the development of molecular imaging probes for GGT are addressed.     

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.     

SNAP-Tag-Targeted MRI-Fluorescent Multimodal Probes

Magnetic resonance imaging (MRI) is a powerful imaging modality, widely employed in research and clinical settings. However, MRI images suffer from low signals and a lack of target specificity. We aimed to develop a multimodal imaging probe to detect targeted cells by MRI and fluorescence microscopy. We synthesized a trifunctional imaging probe consisting of a SNAP-tag substrate for irreversible and specific labelling of cells, cyanine dyes for bright fluorescence, and a chelated Gd^III molecule for enhancing MRI contrast. Our probes exhibit specific and efficient labelling of genetically defined cells (expressing SNAP-tag at their membrane), bright fluorescence and MRI signal. Our synthetic approach provides a versatile platform for the production of multimodal imaging probes, particularly for light microscopy and MRI.