A Bioorthogonal Probe for Multiscale Imaging by 19F-MRI and Raman Microscopy: From Whole Body to Single Cells.

Molecular imaging techniques are essential tools for better investigating biological processes and detecting disease biomarkers with improvement of both diagnosis and therapy monitoring. Often, a single imaging technique is not sufficient to obtain comprehensive information at different levels. Multimodal diagnostic probes are key tools to enable imaging across multiple scales. The direct registration of in vivo imaging markers with ex vivo imaging at the cellular level with a single probe is still challenging. Fluorinated (19F) probes have been increasingly showing promising potentialities for in vivo cell tracking by 19F-MRI. Here we present the unique features of a bioorthogonal 19F-probe that enables direct signal correlation of MRI with Raman imaging. In particular, we reveal the ability of PERFECTA, a superfluorinated molecule, to exhibit a remarkable intense Raman signal distinct from cell and tissue fingerprints. Therefore, PERFECTA combines in a single molecule excellent characteristics for both macroscopic in vivo 19F-MRI, across the whole body, and microscopic imaging at tissue and cellular levels by Raman imaging.

Multicore Liquid Perfluorocarbon-Loaded Multimodal Nanoparticles for Stable Ultrasound and 19F MRI Applied to In Vivo Cell Tracking.

Ultrasound is the most commonly used clinical imaging modality. However, in applications requiring cell-labeling, the large size and short active lifetime of ultrasound contrast agents limit their longitudinal use. Here, 100 nm radius, clinically applicable, polymeric nanoparticles containing a liquid perfluorocarbon, which enhance ultrasound contrast during repeated ultrasound imaging over the course of at least 48 h, are described. The perfluorocarbon enables monitoring the nanoparticles with quantitative 19F magnetic resonance imaging, making these particles effective multimodal imaging agents. Unlike typical core-shell perfluorocarbon-based ultrasound contrast agents, these nanoparticles have an atypical fractal internal structure. The nonvaporizing highly hydrophobic perfluorocarbon forms multiple cores within the polymeric matrix and is, surprisingly, hydrated with water, as determined from small-angle neutron scattering and nuclear magnetic resonance spectroscopy. Finally, the nanoparticles are used to image therapeutic dendritic cells with ultrasound in vivo, as well as with 19F MRI and fluorescence imaging, demonstrating their potential for long-term in vivo multimodal imaging.

Multispectral MRI with Dual Fluorinated Probes to Track Mononuclear Cell Activity in Mice.

Background MRI with fluorine 19 (19F) probes has shown an ability to track immune cell activity with a specific, stable, and quantitative signal. In addition, the chemical shift differences of selected 19F probes make dual-probe imaging possible. To improve 19F MRI sensitivity for dual-probe imaging, optimal fluorine probes are needed. Purpose To develop multispectral 19F MRI to image immune cell activity in vivo using 19F nanoparticles of two distinct fluorocarbons. Materials and Methods Both 19F nanoparticles formulated with two fluorocarbons with distinct resonance frequencies and a high fluorine payload were characterized in terms of size, stability, MR profile, and relaxation times at 7 T. 19F MRI sensitivity was tested on labeling cells both in vitro and in vivo in C57BL/6 mice after conditional ablation of myeloid cells through the inhibition of colony-stimulating factor-1 receptor (CSF1Ri) to monitor the change of immune cells phagocytosis. Fluorine MRI data were acquired at the resonance frequency of each fluorocarbon by using a three-dimensional fast spin-echo sequence. Fluorescent dyes were also inserted into 19F nanoparticles to allow flow-cytometric and confocal microscopy analysis of labeled cells. Fluorine signal-to-noise ratio (SNR) was compared by using two-way repeated measures analysis of variance with Bonferroni post hoc correction. Results Fluorine MRI demonstrated high sensitivity and high specificity in the imaging of mononuclear cells both in vitro and in vivo. In combination with proton MRI, a map of 19F nuclei from each fluorocarbon was obtained without overlaps or artifacts. In vitro cell viability was unchanged, and 8000 cells with a high SNR (>8) were detected. In vivo high fluorine signal was observed in the bone marrow (SNR > 15) immediately after CSF1Ri treatment interruption, which correlated with high uptake by neutrophils and monocytes at flow cytometry. Conclusion By assessing in vivo MRI of mononuclear cell phagocytic ability with 19F nanoparticles, MRI with dual 19F probes can effectively track immune cell activity in combination with current MRI protocols. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Bulte in this issue.

The scope of nanoparticle therapies for future metastatic melanoma treatment.

Metastatic melanoma is a highly aggressive malignancy that has traditionally been very difficult to treat. However, after decades of basic research into the signal transduction pathways that promote cancer cell survival, chemoresistance, growth, and crosstalk with the immune system, targeted therapies have now been developed that offer improved survival for patients with metastatic melanoma. Some of the most promising therapies that have been developed include ipilimumab, an anti-cytotoxic T lymphocyte antigen 4 antibody that enhances T-cell activity in the tumour, and selective BRAF inhibitors, such as vemurafenib that blocks tumour cell proliferation in patients with activating BRAF mutations. Although these treatments offer substantial hope for patients, they are not without their drawbacks, which include adverse side-effects, drug resistance, and eventual relapse. Nanotherapeutics holds significant promise to circumvent these shortcomings and has the additional advantage of potentially functioning as a diagnostic device. We will discuss the scope of the use of such multimodal nanoparticles for melanoma treatment and ask whether such particles can offer patients with metastatic melanoma improved prognoses for the future.

Next-generation agents for fluorescence-guided glioblastoma surgery.

Glioblastoma is a fast-growing and aggressive form of brain cancer. Even with maximal treatment, patients show a low median survival and are often subjected to a high recurrence incidence. The currently available treatments require multimodal management, including maximal safe surgical resection, followed by radiation and chemotherapy. Because of the infiltrative glioblastoma nature, intraoperative differentiation of cancer tissue from normal brain parenchyma is very challenging, and this accounts for the low rate of complete tumor resection. For these reasons, clinicians have increasingly used various intraoperative adjuncts to improve surgical results, such as fluorescent agents. However, most of the existing fluorophores show several limitations such as poor selectivity, photostability, photosensitization and high costs. This could limit their application to successfully improve glioblastoma resection. In the present perspective, we highlight the possibility to develop next-generation fluorescent tools able to more selectively label cancer cells during surgical resection.

FM19G11-loaded nanoparticles modulate energetic status and production of reactive oxygen species in myoblasts from ALS mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Considerable evidence indicates that early skeletal muscle atrophy plays a crucial role in the disease pathogenesis, leading to an altered muscle-motor neuron crosstalk that, in turn, may contribute to motor neuron degeneration. Currently, there is no effective treatment for ALS, highlighting the need to dig deeper into the pathological mechanisms for developing innovative therapeutic strategies. FM19G11 is a novel drug able to modulate the global cellular metabolism, but its effects on ALS skeletal muscle atrophy and mitochondrial metabolism have never been evaluated, yet. This study investigated whether FM19G11-loaded nanoparticles (NPs) may affect the bioenergetic status in myoblasts isolated from G93A-SOD1 mice at different disease stages. We found that FM19G1-loaded NP treatment was able to increase transcriptional levels of Akt1, Akt3, Mef2a, Mef2c and Ucp2, which are key genes associated with cell proliferation (Akt1, Akt3), muscle differentiation (Mef2c), and mitochondrial activity (Ucp2), in G93A-SOD1 myoblasts. These cells also showed a significant reduction of mitochondrial area and networks, in addition to decreased ROS production after treatment with FM19G11-loaded NPs, suggesting a ROS clearance upon the amelioration of mitochondrial dynamics. Our overall findings demonstrate a significant impact of FM19G11-loaded NPs on muscle cell function and bioenergetic status in G93A-SOD1 myoblasts, thus promising to open new avenues towards possible adoption of FM19G11-based nanotherapies to slow muscle degeneration in the frame of ALS and muscle disorders.

Interaction of polymers with bile salts - Impact on solubilisation and absorption of poorly water-soluble drugs.

Formulating poorly soluble drugs with polymers in the form of solid dispersions has been widely used for improving drug dissolution. Endogenous surface-active species present in the gut, such as bile salts, lecithin and other phospholipids, have been shown to play a key role in facilitating lipids and poorly soluble drugs solubilisation in the gut. In this study, we examined the possible occurrence of interactions between a model bile salt, sodium taurocholate (NaTC), and model spray dried solid dispersions comprising piroxicam and Hydroxypropyl Methylcellulose (HPMC), a commonly used hydrophilic polymer for solid dispersion preparation. Solubility measurements revealed the good solubilisation effect of NaTC on the crystalline drug, which was enhanced by the addition of HPMC, and further boosted by the drug formulation into solid dispersion. The colloidal behaviour of the solid dispersions upon dissolution in biorelevant media, with and without NaTC, revealed the formation of NaTC-HPMC complexes and other mixed colloidal species. Cellular level drug absorption studies obtained using Caco-2 monolayers confirmed that the combination of drug being delivered by solid dispersion and the presence of bile salt and lecithin significantly contributed to the improved drug absorption. Together with the role of NaTC-HPMC complexes in assisting the drug solubilisation, our results also highlight the complex interplay between bile salts, excipients and drug absorption.

Pivotal role of the protein corona in the cell uptake of fluorinated nanoparticles with increased sensitivity for 19F-MR imaging.

In vivo cell tracking by non-invasive imaging technologies is needed to accelerate the clinical translation of innovative cell-based therapies. In this regard, 19F-MRI has recently gained increased attention for unbiased localization of labeled cells over time. To push forward the use of 19F-MRI for cell tracking, the development of highly performant 19F-probes is required. PLGA-based NPs containing PERFECTA, a multibranched superfluorinated molecule with an optimal MRI profile thanks to its 36 magnetically equivalent fluorine atoms, are promising 19F-MRI probes. In this work we demonstrate the importance of the surface functionalization of these NPs in relation to their interaction with the biological environment, stressing the pivotal role of the formation of the protein corona (PC) in their cellular labelling efficacy. In particular, our studies showed that the formation of PC NPs strongly promotes the cellular internalization of these NPs in microglia cells. We advocate that the formation of PC NPs in the culture medium can be a key element to be used for the optimization of cell labelling with a considerable increase of the detection sensitivity by 19F-MRI.

Surface coatings shape the protein corona of SPIONs with relevance to their application in vivo.

Superparamagnetic iron oxide nanoparticles (SPIONs) have proved their use in many biomedical applications, such as drug delivery, hyperthermia, and MRI (magnetic resonance imaging) contrast agents. Due to their instability in fluids, several surface coatings have been used to both stabilize and tune the properties of these nanoparticles (NPs) according to their applications. These coatings will strongly modify their surface properties and influence their interaction with the environment proteins in a relevant biological medium with a clear impact on their function. It is well-accepted that a protein corona is immediately formed when nanoparticles come in contact with a biological milieu, and the emergent bionano interface represents the biological identity of the particles. Here, we investigate how a different coating on the same magnetic core can influence the protein corona composition and structure with clear relevance to application of these NPs in medicine. In particular, we have studied the structure and composition of the protein corona-SPION complexes of magnetite nanoparticles stabilized with citric acid, poly(acrylic acid), or double layer oleic acid by a range of approaches, including dynamic light scattering, nanoparticle tracking analysis, differential centrifugal sedimentation, infrared spectroscopy, 1-D SDS gel electrophoresis, and mass spectroscopy.

High-resolution crystal structure of a 20 kDa superfluorinated gold nanocluster.

Crystallization of atomically precise nanoclusters is gaining increasing attention, due to the opportunity of elucidating both intracluster and intercluster packing modes, and exploiting the functionality of the resulting highly pure crystallized materials. Herein, we report the design and single-crystal X-ray structure of a superfluorinated 20 kDa gold nanocluster, with an Au25 core coated by a shell of multi-branched highly fluorinated thiols (SF27) resulting in almost 500 fluorine atoms, i.e., ([Au25(SF27)18]0). The cluster shows a switchable solubility in the fluorous phase. X-ray analysis and computational studies reveal the key role of both intracluster and intercluster F···F contacts in driving [Au25(SF27)18]0 crystal packing and stabilization, highlighting the ability of multi-branched fluorinated thiols to endow atomically precise nanoclusters with remarkable crystallogenic behavior.

Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles.

It is now clearly emerging that besides size and shape, the other primary defining element of nanoscale objects in biological media is their long-lived protein ("hard") corona. This corona may be expressed as a durable, stabilizing coating of the bare surface of nanoparticle (NP) monomers, or it may be reflected in different subpopulations of particle assemblies, each presenting a durable protein coating. Using the approach and concepts of physical chemistry, we relate studies on the composition of the protein corona at different plasma concentrations with structural data on the complexes both in situ and free from excess plasma. This enables a high degree of confidence in the meaning of the hard protein corona in a biological context. Here, we present the protein adsorption for two compositionally different NPs, namely sulfonated polystyrene and silica NPs. NP-protein complexes are characterized by differential centrifugal sedimentation, dynamic light scattering, and zeta-potential both in situ and once isolated from plasma as a function of the protein/NP surface area ratio. We then introduce a semiquantitative determination of their hard corona composition using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray liquid chromatography mass spectrometry, which allows us to follow the total binding isotherms for the particles, identifying simultaneously the nature and amount of the most relevant proteins as a function of the plasma concentration. We find that the hard corona can evolve quite significantly as one passes from protein concentrations appropriate to in vitro cell studies to those present in in vivo studies, which has deep implications for in vitro-in vivo extrapolations and will require some consideration in the future.

What the cell "sees" in bionanoscience.

What the biological cell, organ, or barrier actually "sees" when interacting with a nanoparticle dispersed in a biological medium likely matters more than the bare material properties of the particle itself. Typically the bare surface of the particle is covered by several biomolecules, including a select group of proteins drawn from the biological medium. Here, we apply several different methodologies, in a time-resolved manner, to follow the lifetime of such biomolecular "coronas" both in situ and isolated from the excess plasma. We find that such particle-biomolecule complexes can be physically isolated from the surrounding medium and studied in some detail, without altering their structure. For several nanomaterial types, we find that blood plasma-derived coronas are sufficiently long-lived that they, rather than the nanomaterial surface, are likely to be what the cell sees. From fundamental science to regulatory safety, current efforts to classify the biological impacts of nanomaterials (currently according to bare material type and bare surface properties) may be assisted by the methodology and understanding reported here.

In Situ Generation of Chiroptically-Active Gold-Peptide Superstructures Promoted by Iodination.

Peptide-mediated routes to the synthesis of plasmonic nanoparticles have been drawing increasing attention for the development of chiroptically active nanoscale architectures. However, designing a multifunctional peptide able to drive the formation of structurally defined nanomaterials endowed with specific functionalities is still challenging. In this work, iodination has been devised as a strategy to strengthen Au-reduction capability of the amyloidogenic peptide DFNKF and combine it with its distinctive self-assembly features. Thanks to the Au-mediated C-I activation on the phenylalanine iodobenzenes, the peptides yield efficient Au-reduction ability promoting the synthesis of Au nanoparticles, and simultaneously working as templates for their spontaneous self-assembly into spherical superstructures endowed with chiroptical activities. The reaction occurs in situ through a one-pot process in aqueous media. The generality of this approach has been demonstrated using an iodinated derivative of the peptide KLVFF, which also showed reducing and templating abilities forming chiroptically active helical superstructures decorated with Au nanoparticles.

DNA closed nanostructures: a structural and Monte Carlo simulation study.

DNA nanoconstructs are obtained in solution by using six unique 42-mer DNA oligonucleotides, whose sequences have been designed to form a pseudohexagonal structure. The required flexibility is provided by the insertion of two non-base-paired thymines in the middle of each sequence that work as flexible hinges and constitute the corners of the nanostructure when formed. We show that hexagonally shaped nanostructures of about 7 nm diameter and their corresponding linear open constructs are formed by self-assembly of the specifically designed linear oligonucleotides. The structural and dynamical characterization of the nanostructure is obtained in situ for the first time by using dynamic light scattering (DLS), a noninvasive method that provides a fast dynamic and structural analysis and allows the characterization of the different synthetic DNA nanoconstructs in solution. A validation of the LS results is obtained through Monte Carlo (MC) simulations and atomic force microscopy (AFM). In particular, a mesoscale molecular model for DNA, developed by Knotts et al., is exploited to perform MC simulations and to obtain information about the conformations as well as the conformational flexibilities of these nanostructures, while AFM provides a very detailed particle analysis that yields an estimation of the particle size and size distribution. The structural features obtained by MC and AFM are in good agreement with DLS, showing that DLS is a fast and reliable tool for characterization of DNA nanostructures in solution.

Phospholipid membranes decorated by cholesterol-based oligonucleotides as soft hybrid nanostructures.

DNA monomers and oligomers are currently showing great promise as building blocks for supramolecular arrays that can self-assemble in a fashion preprogrammed by the base pairing code. The design and build-up of hybrid DNA/amphiphilic self-assemblies can expand the range of possible architectures and enhance the selectivity toward a well-specified geometry. We report on the self-assembly properties in aqueous solution of a cholesteryl-tetraethylenglycol single stranded 18-mer oligonucleotide (ON 1TEG-Chol) and on its spontaneous insertion in fluid phospholipid membranes. Up to 500 units of these lipophilic ss-oligonucleotides can be incorporated in the outer leaflet of 350 A radius POPC vesicle. The insertion and hybridization with the complementary oligonucleotide are monitored through light scattering as an increase of hydrodynamic thickness, which is interpreted in terms of average distance between anchoring sites. The conformation of the ss-oligonucleotidic portion is strongly dependent on surface coverage, passing from a quasi-random coil to a more rigid configuration, as concentration increases. Interestingly, conformational details affect in a straightforward fashion the hybridization kinetics. Liposomes with single- and double-strand decorations remain stable within the experimental time window (about one week). The structure represents an example of successful and stable amphiphile/DNA supramolecular hybrid, where a DNA guest is held in a membrane by hydrophobic interactions. The lipophilic oligonucleotide under investigation is therefore a suitable building block that can effectively serve as a hydrophobic anchor in the fluid bilayer to assemble supramolecular constructs based on the DNA digital code.

Amphiphilic self-assemblies decorated by nucleobases.

Phosphatidyl-nucleosides are a class of functional amphiphiles, where a nucleic acid monomer is conjugated to a lipid skeleton. These derivatives self-organize in aqueous solution as assemblies of various size, shape, and interfacial curvature. This paper presents a comparison of the aggregation behavior of different 1-R,2-R-sn-glycero-3-phosphatidyl-nucleosides, where R = 8 (DiC8PN) or R = 12 (DLPN) and N is either adenosine (a purine) or uridine (a pyrimidine), a complementary pair in RNA. Surface tension, small angle neutron scattering, cryo-TEM, and circular dichroism are used to highlight and distinguish the impact of the hydrophobic assembler and of the base substitution on the solution phase behavior. Our main conclusion is that the nucleic functionalization provides an additional parameter to control self-assembly through specific interactions among the polar heads. Further nonideal effects are induced by mixing nucleolipids with complementary base substitution. We show that these contributions alter the aggregation thresholds and modulate properties of the aggregates on the mesoscale.

Combining Cytotoxicity Assessment and Xenopus laevis Phenotypic Abnormality Assay as a Predictor of Nanomaterial Safety.

The African clawed frog, Xenopus laevis, has been used as an efficient pre-clinical screening tool to predict drug safety during the early stages of the drug discovery process. X. laevis is a relatively inexpensive model that can be used in whole organism high-throughput assays whilst maintaining a high degree of homology to the higher vertebrate models often used in scientific research. Despite an ever-increasing volume of biomedical nanoparticles (NPs) in development, their unique physico-chemical properties challenge the use of standard toxicology assays. Here, we present a protocol that directly compares the sensitivity of X. laevis development as a tool to assess potential NP toxicity by observation of embryo phenotypic abnormalities/lethality after NP exposure, to in vitro cytotoxicity obtained using mammalian cell lines. In combination with conventional cytotoxicity assays, the X. laevis phenotypic assay provides accurate data to efficiently assess the safety of novel biomedical NPs. © 2017 by John Wiley & Sons, Inc.

Crystallographic insights into the structural aspects of thioctic acid based halogen-bond donor for the functionalization of gold nanoparticles.

The synthesis and self-assembly capabilities of a new halogen-bond donor ligand, 2,3,5,6-tetrafluoro-4-iodophenyl 5-(1,2-dithiolan-3-yl)pentanoate (1), are reported. The crystal structure of ligand (1) and the formation of a cocrystal with 1,2-di(4-pyridyl)ethylene, (1)·(2), both show halogen bonds involving the 4-iodotetrafluorobenzene moiety. Ligand (1), being a self-complementary unit, forms an infinite halogen-bonded chain driven by the S...I synthon, while the cocrystal (1)·(2) self-assembles into a discrete trimeric entity driven by the N...I synthon. Ligand (1) was also successfully used to functionalize the surface of gold nanoparticles, AuNP-(1). Experiments on the dispersibility profile of AuNP-(1) demonstrated the potential of halogen bonding in facilitating the dispersion of modified NPs with halogen-bond donors in pyridine.