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.

Design of fluorinated stealth poly(ε-caprolactone) nanocarriers.

The covalent functionalization of polymers with fluorinated moieties represents a promising strategy for the development of multimodal systems. Moreover, polymer fluorination often endows the resulting nanocarriers with improved colloidal stability in the biological environment. In this work, we developed fluorinated pegylated (PEG) biodegradable poly(ε-caprolactone) (PCL) drug nanocarriers showing both high colloidal stability and stealth properties, as well as being (19F)-Nuclear Magnetic Resonance (NMR) detectable. The optimized nanocarriers were obtained mixing a PEG-PCL block copolymer with a nonafluoro-functionalized PCL polymer. The role of PEGylation and fluorination on self-assembly and colloidal behavior of the obtained nanoparticles (NPs) was investigated, as well as their respective role on stealth properties and colloidal stability. To prove the feasibility of the developed NPs as potential 19F NMR detectable drug delivery systems, a hydrophobic drug was successfully encapsulated, and the maintenance of the relevant 19F NMR properties evaluated. Drug-loaded fluorinated NPs still retained a sharp and intense 19F NMR signal and good relaxivity parameters (i.e., T1 and T2 relaxation times) in water, which were not impaired by drug encapsulation.

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.

Superfluorinated Extracellular Vesicles for In Vivo Imaging by 19F-MRI.

Extracellular vesicles (EVs) play a crucial role in cell-to-cell communication and have great potential as efficient delivery vectors. However, a better understanding of EV in vivo behavior is hampered by the limitations of current imaging tools. In addition, chemical labels present the risk of altering the EV membrane features and, thus, in vivo behavior. 19F-MRI is a safe bioimaging technique providing selective images of exogenous probes. Here, we present the first example of fluorinated EVs containing PERFECTA, a branched molecule with 36 magnetically equivalent 19F atoms. A PERFECTA emulsion is given to the cells, and PERFECTA-containing EVs are naturally produced. PERFECTA-EVs maintain the physicochemical features, morphology, and biological fingerprint as native EVs but exhibit an intense 19F-NMR signal and excellent 19F relaxation times. In vivo 19F-MRI and tumor-targeting capabilities of stem cell-derived PERFECTA-EVs are also proved. We propose PERFECTA-EVs as promising biohybrids for imaging biodistribution and delivery of EVs throughout the body.

Optimization of superfluorinated PLGA nanoparticles for enhanced cell labelling and detection by 19F-MRI.

Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is an emergent imaging technique for molecular imaging and cell tracking. Lack of intrinsic 19F signals in tissues allows unambiguous in vivo detection of exogenous fluorinated probes, complementary to the anatomical and multiparametric information obtained by standard 1H-MRI. However, the intrinsic low sensitivity of MRI technique requires the need of designing increasingly effective fluorinated tracers. PERFECTA, with its 36 magnetically equivalent 19F atoms and a designed branched molecular structure, represents an excellent superfluorinated tracer. In this paper, we report the development of PERFECTA loaded PLGA NPs stabilized by different coatings as promising 19F-MRI probes. The results clearly show the optimal cellular uptake of the produced colloidally stable PERFECTA loaded PLGA NPs without impact on cells viability. Importantly, NPs stabilization with the anionic surfactant sodium cholate (NaC) clearly enhances NPs internalization within cells with respect to PVA-coated NPs. Moreover, the optimized NPs are characterized by shorter T1 relaxation times with respect to other PERFECTA formulations that would allow the increase of 19F-MRI sensitivity with fast imaging acquisitions.

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.

Non-invasive visual evoked potentials to assess optic nerve involvement in the dark agouti rat model of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein.

Experimental autoimmune encephalomyelitis (EAE) is the primary disease model of multiple sclerosis (MS), one of the most diffused neurological diseases characterized by fatigue, muscle weakness, vision loss, anxiety and depression. EAE can be induced through injection of myelin peptides to susceptible mouse or rat strains. In particular, EAE elicited by the autoimmune reaction against myelin oligodendrocyte glycoprotein (MOG) presents the common features of human MS: inflammation, demyelination and axonal loss. Optic neuritis affects visual pathways in both MS and in several EAE models. Neurophysiological evaluation through visual evoked potential (VEP) recording is useful to check visual pathway dysfunctions and to test the efficacy of innovative treatments against optic neuritis. For this purpose, we investigate the extent of VEP abnormalities in the dark agouti (DA) rat immunized with MOG, which develops a relapsing-remitting disease course. Together with the detection of motor signs, we acquired VEPs during both early and late stages of EAE, taking advantage of a non-invasive recording procedure that allows long follow-up studies. The validation of VEP outcomes was determined by comparison with ON histopathology, aimed at revealing inflammation, demyelination and nerve fiber loss. Our results indicate that the first VEP latency delay in MOG-EAE DA rats appeared before motor deficits and were mainly related to an inflammatory state. Subsequent VEP delays, detected during relapsing EAE phases, were associated with a combination of inflammation, demyelination and axonal loss. Moreover, DA rats with atypical EAE clinical course tested at extremely late time points, manifested abnormal VEPs although motor signs were mild. Overall, our data demonstrated that non-invasive VEPs are a powerful tool to detect visual involvement at different stages of EAE, prompting their validation as biomarkers to test novel treatments against MS optic neuritis.

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.