Glycan-Gold Nanoparticles as Multifunctional Probes for Multivalent Lectin-Carbohydrate Binding: Implications for Blocking Virus Infection and Nanoparticle Assembly.

Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.

10-Plex Digital Polymerase Chain Reaction with Four-Color Melting Curve Analysis for Simultaneous KRAS and BRAF Genotyping.

Digital PCR (dPCR) is a promising method for performing liquid biopsies that quantifies nucleic acids more sensitively than real-time PCR. However, dPCR shows large fluctuations in the fluorescence intensity of droplets or wells due to insufficient PCR amplification in the small partitions, limiting the multiplexing capability of using the fluorescence intensity. In this study, we propose a measurement method that combines dPCR with melting curve analysis for highly multiplexed genotyping. A sample was digitized into a silicon chip with up to 2 × 104 wells in which asymmetric PCR was performed to obtain more single-stranded amplicons that were complementary to molecular beacon probes. Fluorescence images were captured while controlling the temperature of the chip, and the melting curve was measured for each well. Then, genotyping was performed by using the fluorescence intensity, the dye color of the probe, and the melting temperature (Tm). Because the Tm of the PCR products is not highly dependent on the amplification efficiency of PCR, genotyping accuracy is improved by using Tm values, enabling highly multiplexed genotyping. The concept was confirmed by simultaneously identifying wild-type KRAS, BRAF, and eight mutants of these genes (G12D, G12R, G12V, G13D, G12A, G12C, G12S, and V600E) through four-color melting curve analysis. To the best of our knowledge, this is the first demonstration of the genotyping of 10 DNA groups including single mutations of cancer-related genes by combining dPCR with four-color melting curve analysis.

Molecular Imaging of Fluorinated Probes for Tau Protein and Amyloid-ß Detection.

Alzheimer's disease (AD) is the most common form of dementia and results in progressive neurodegeneration. The incidence rate of AD is increasing, creating a major public health issue. AD is characterized by neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein and senile plaques composed of amyloid-ß (Aß). Currently, a definitive diagnosis of AD is accomplished post-mortem. Thus, the use of molecular probes that are able to selectively bind to NFTs or Aß can be valuable tools for the accurate and early diagnosis of AD. The aim of this review is to summarize and highlight fluorinated molecular probes that can be used for molecular imaging to detect either NFTs or Aß. Specifically, fluorinated molecular probes used in conjunction with 19F MRI, PET, and fluorescence imaging will be explored.

Targeted, Stimuli-Responsive, and Theranostic 19F Magnetic Resonance Imaging Probes.

Unlike conventional 1H magnetic resonance imaging (MRI), 19F MRI features unambiguous detection of fluorine spins due to negligible background signals. Therefore, it is considered a promising noninvasive and selective imaging method for the diagnosis of cancers and other diseases. For 19F MRI, fluorine-rich molecules such as perfluorocarbons (PFC) have been formulated into nanoemulsions and used as its tracer agent. Along with advancements in other types of nanoparticles as targeted theranostics and stimuli-triggered probes and combined with the advantages of 19F MRI, PFC nanoemulsions are being empowered with these additional functionalities and becoming a promising theranostic platform. In this Review, we provide an overview of fluorine-based materials for sensitive 19F MRI of biological and pathological conditions. In particular, we describe designs and applications of recently reported stimuli-responsive and theranostic 19F MRI probes. Finally, challenges and future perspectives regarding the further development of 19F MRI probes for their clinical applications are described.

Cyclotides: Overview and Biotechnological Applications.

Cyclotides are globular microproteins with a unique head-to-tail cyclized backbone, stabilized by three disulfide bonds forming a cystine knot. This unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to chemical, thermal, and biological degradation compared to other peptides of similar size. In addition, cyclotides have been shown to be highly tolerant to sequence variability, aside from the conserved residues forming the cystine knot. Cyclotides can also cross cellular membranes and are able to modulate intracellular protein-protein interactions, both in vitro and in vivo. All of these features make cyclotides highly promising as leads or frameworks for the design of peptide-based diagnostic and therapeutic tools. This article provides an overview on cyclotides and their applications as molecular imaging agents and peptide-based therapeutics.

Nanofabricated Neural Probes for Dense 3-D Recordings of Brain Activity.

Computations in brain circuits involve the coordinated activation of large populations of neurons distributed across brain areas. However, monitoring neuronal activity in the brain of intact animals with high temporal and spatial resolution has remained a technological challenge. Here we address this challenge by developing dense, three-dimensional (3-D) electrode arrays for electrophysiology. The 3-D arrays constitute the front-end of a modular and configurable system architecture that enables monitoring neuronal activity with unprecedented scale and resolution.

Combating neurodegenerative disease with chemical probes and model systems.

The disheartening results of recent clinical trials for neurodegenerative disease (ND) therapeutics underscore the need for a more comprehensive understanding of the underlying disease biology before effective therapies can be devised. One hallmark of many NDs is a disruption in protein homeostasis. Therefore, investigating the role of protein homeostasis in these diseases is central to delineating their underlying pathobiology. Here, we review the seminal role that chemical biology has played in furthering the research on and treatment of dysfunctional protein homeostasis in NDs. We also discuss the vital and predictive role of model systems in identifying conserved homeostasis pathways and genes therein that are altered in neurodegeneration. Integrating approaches from chemical biology with the use of model systems yields a powerful toolkit with which to unravel the complexities of ND biology.

Bifunctional molecular probe targeting tumor PD-L1 enhances anti-tumor efficacy by promoting ferroptosis in lung cancer mouse model.

PURPOSE: Immune checkpoint inhibitors (ICIs) targeting tumor-specific PD-1/PD-L1 significantly improve the overall survival rate of patients with advanced cancer by reactivating the immune system to attack cancer cells. To explore their tumor killing effect, we used the radionuclide iodine-131 (131I) to label the anti-PD-L1 antibody Atezolizumab (131I-PD-L1 mAb). METHOD: We prepared the radioimmunoassay molecular probe 131I-PD-L1 mAb by the chloramine-T method and evaluated its affinity using Lewis lung cancer (LLC) cells. The uptake of 131I-PD-L1 mAb by transplanted tumors was examined through SPECT and its in vivo distribution. We then compared the in vitro and in vivo anti-tumor efficacy of groups treated with control, PD-L1 mAb, 131I-PD-L1 mAb, and 131I-PD-L1 mAb + PD-L1 mAb combined treatment. We performed H&E staining to examine the changes in tumor, as well as the damage in major tissues and organs caused by potential side effects. The anti-tumor mechanism of 131I-PD-L1 mAb was analyzed by Western blot, RT-qPCR and immunohistochemistry (IHC). RESULT: 131I-PD-L1 mAb was highly stable and specific, and easily penetrated into tumor. 131I-PD-L1 mAb suppressed cancer cell proliferation in vitro, and inhibited tumor growth in vivo by inducing ferroptosis, thus prolonging the survival of experimental animals while demonstrating biological safety. CONCLUSION: Therefore, our study suggested that 131I-PD-L1 mAb affected the expression of tumor-related factors through ß-rays and thus promoted ferroptosis in tumor. Combined treatment showed better anti-tumor effect compared to single ICI treatment.

Ruthenium(II) polypyridyl complexes and DNA--from structural probes to cellular imaging and therapeutics.

In the last few decades, coordination complexes based on d(6) metal centres and polypyridyl ligand architectures been developed as structure- and site-specific reversible DNA binding agents. Due to their attractive photophysical properties, much of this research has focused on complexes based on ruthenium(II) centres and, more recently, attention has turned to the use of these complexes in biological contexts. As the rules that govern the cellular uptake and cellular localisation of such systems are determined they are finding numerous applications ranging from imaging to therapeutics. This review illustrates how the interdisciplinary nature of this research-which takes in synthetic chemistry, biophysical and in cellulo studies-makes this an exciting area in which an array of further applications are likely to emerge.

Upconversion nanophosphors for small-animal imaging.

Rare-earth upconversion nanophosphors (UCNPs), when excited by continuous-wave near-infrared light, exhibit a unique narrow photoluminescence with higher energy. Such special upconversion luminescence makes UCNPs promising as bioimaging probes with attractive features, such as no auto-fluorescence from biological samples and a large penetration depth. As a result, UCNPs have emerged as novel imaging agents for small animals. In this critical review, recent reports regarding the synthesis of water-soluble UCNPs and their surface modification and bioconjugation chemistry are summarized. The applications of UCNPs for small-animal imaging, including tumor-targeted imaging, lymphatic imaging, vascular imaging and cell tracking are reviewed in detail. The exploration of UCNPs as multifunctional nanoscale carriers for integrated imaging and therapy is also presented. The biodistribution and toxicology of UCNPs are further described. Finally, we discuss the challenges and opportunities in the development of UCNP-based nanoplatforms for small-animal imaging (276 references).

Theranostic nanoshells: from probe design to imaging and treatment of cancer.

Recent advances in nanoscience and biomedicine have expanded our ability to design and construct multifunctional nanoparticles that combine targeting, therapeutic, and diagnostic functions within a single nanoscale complex. The theranostic capabilities of gold nanoshells, spherical nanoparticles with silica cores and gold shells, have attracted tremendous attention over the past decade as nanoshells have emerged as a promising tool for cancer therapy and bioimaging enhancement. This Account examines the design and synthesis of nanoshell-based theranostic agents, their plasmon-derived optical properties, and their corresponding applications. We discuss the design and preparation of nanoshell complexes and their ability to enhance the photoluminescence of fluorophores while maintaining their properties as MR contrast agents. In this Account, we discuss the underlying physical principles that contribute to the photothermal response of nanoshells. We then elucidate the photophysical processes that induce nanoshells to enhance the fluorescence of weak near-infrared fluorophores. Nanoshells illuminated with resonant light are either strong optical absorbers or scatterers, properties that give rise to their unique capabilities. These physical processes have been harnessed to visualize and eliminate cancer cells. We describe the application of nanoshells as a contrast agent for optical coherence tomography of breast carcinoma cells in vivo. Our recent studies examine nanoshells as a multimodal theranostic probe, using these nanoparticles for near-infrared fluorescence and magnetic resonance imaging (MRI) and for the photothermal ablation of cancer cells. Multimodal nanoshells show theranostic potential for imaging subcutaneous breast cancer tumors in animal models and the distribution of tumors in various tissues. Nanoshells also show promise as light-triggered gene therapy vectors, adding temporal control to the spatial control characteristic of nanoparticle-based gene therapy approaches. We describe the fabrication of DNA-conjugated nanoshell complexes and compare the efficiency of light-induced and thermally-induced release of DNA. Double-stranded DNA nanoshells also provide a way to deliver small molecules into cells: we describe the delivery and light-triggered release of DAPI (4',6-diamidino-2-phenylindole), a dye molecule used to stain DNA in the nuclei of cells.

New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation.

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a ß(-) emitter such as (90)Y with an optical imager.

Peptide-based molecular beacons for cancer imaging and therapy.

Peptide-based molecular beacons are Förster resonance energy transfer-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets and thus are useful tools for in vivo cancer imaging. With our increasing knowledge about human genome in health and disease, peptide-based "smart" probes are continually developed for in vivo optical imaging of specific molecular targets, biological pathways and cancer progression and diagnosis. A class of fluorescent photosensitizers further extends the application of peptide beacons to cancer therapeutics. This review highlights the applications of peptide beacons in cancer imaging, the simultaneous treatment and response monitoring and smart therapeutics with a focus on recent improvements in the design of these probes.

Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system.

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.

Surface Probe Linker with Tandem Anti-Fouling Properties for Application in Biosensor Technology.

This paper describes the anti-fouling capability of the novel monolayer-forming surface linker 3-(3-(trichlorosilylpropyloxy) propanoyl chloride (MEG-Cl). This compound was successfully attached to quartz crystal surfaces which are employed in an electromagnetic piezoelectric acoustic sensor (EMPAS) configuration. The MEG-Cl coated surface was both employed with Ni-NTA for the binding of recombinant proteins and for the tandem property of the avoidance of fouling from serum and milk. The MEG-Cl coated surfaces were found to provide a large degree of anti-fouling on the EMPAS device, and were comparable to previously studied MEG-OH surfaces. Importantly, the monolayer continued to provide anti-fouling capability to the biosensor following extension with Ni-NTA in place. Accordingly, this surface linker provides an attractive system for use in biosensor technology in terms of both its anti-fouling and linking properties.

Recent advances in molecular imaging of atherosclerotic plaques and thrombosis.

As the complications of atherosclerosis such as myocardial infarction and stroke are still one of the leading causes of mortality worldwide, the development of new diagnostic tools for the early detection of plaque instability and thrombosis is urgently needed. Advanced molecular imaging probes based on functional nanomaterials in combination with cutting edge imaging techniques are now paving the way for novel and unique approaches to monitor the inflammatory progress in atherosclerosis. This review focuses on the development of various molecular probes for the diagnosis of plaques and thrombosis in atherosclerosis, along with perspectives of their diagnostic applications in cardiovascular diseases. Specifically, we summarize the biological targets that can be used for atherosclerosis and thrombosis imaging. Then we describe the emerging molecular imaging techniques based on the utilization of engineered nanoprobes together with their challenges in clinical translation.

Nanoparticle-based Cell Trackers for Biomedical Applications.

The continuous or real-time tracking of biological processes using biocompatible contrast agents over a certain period of time is vital for precise diagnosis and treatment, such as monitoring tissue regeneration after stem cell transplantation, understanding the genesis, development, invasion and metastasis of cancer and so on. The rationally designed nanoparticles, including aggregation-induced emission (AIE) dots, inorganic quantum dots (QDs), nanodiamonds, superparamagnetic iron oxide nanoparticles (SPIONs), and semiconducting polymer nanoparticles (SPNs), have been explored to meet this urgent need. In this review, the development and application of these nanoparticle-based cell trackers for a variety of imaging technologies, including fluorescence imaging, photoacoustic imaging, magnetic resonance imaging, magnetic particle imaging, positron emission tomography and single photon emission computing tomography are discussed in detail. Moreover, the further therapeutic treatments using multi-functional trackers endowed with photodynamic and photothermal modalities are also introduced to provide a comprehensive perspective in this promising research field.

Progress in Understanding Ferroptosis and Challenges in Its Targeting for Therapeutic Benefit.

Ferroptosis is an iron-dependent cell-death modality driven by oxidative phospholipid damage. In contrast to apoptosis, which enables organisms to eliminate targeted cells purposefully at specific times, ferroptosis appears to be a vulnerability of cells that otherwise use high levels of polyunsaturated lipids to their advantage. Cells in this high polyunsaturated lipid state generally have safeguards that mitigate ferroptotic risk. Since its recognition, ferroptosis has been implicated in degenerative diseases in tissues including kidney and brain, and is a targetable vulnerability in multiple cancers-each likely characterized by the high polyunsaturated lipid state with insufficient or overwhelmed ferroptotic safeguards. In this Perspective, we present progress toward defining the essential roles and key mediators of lipid peroxidation and ferroptosis in disease contexts. Moreover, we discuss gaps in our understanding of ferroptosis and list key challenges that have thus far limited the full potential of targeting ferroptosis for improving human health.

Chemical biology--understanding biology and advancing therapy.

Chemical biology is a new academic discipline whose goals are to understand biological systems using chemistry and chemical compounds. In addition to serving as important biological probes, some of these small molecules could be useful therapeutically. This review will discuss how advances in chemical biology by academics can aid in the discovery of new anti-cancer agents. Specifically, novel molecules can be useful tools to validate a potential target in a disease site and help provide the rationale for a clinical trial with a related molecule developed by industry. These novel molecules can also be developed for clinical use, although obstacles to this approach are recognized. Finally, this review discusses the opportunities to identify off-patent drugs with previously unrecognized anti-cancer activity and how the prior data on the drug would permit it to be rapidly repurposed.