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

Dose-Related Effects of Repeated ETC-216 (Recombinant Apolipoprotein A-IMilano/1-Palmitoyl-2-Oleoyl Phosphatidylcholine Complexes) Administrations on Rabbit Lipid-Rich Soft Plaques In Vivo Assessment by Intravascular Ultrasound and Magnetic Resonance Imaging

ObjectivesThis study sought to evaluate in vivo the minimal dose of apolipoprotein (apo) A-IMilano phospholipid complex (recombinant apoA-IMilano and 1-palmitoyl-2-oleoyl phosphatidylcholine complexes [ETC-216]) able to induce atherosclerosis regression in a rabbit model of lipid-rich plaques.BackgroundA single high dose of recombinant apoA-IMilano has promoted atherosclerosis regression in animal models. More recently, regression of atherosclerosis was achieved in coronary patients by repeated infusions of ETC-216.MethodsThirty-six rabbits underwent perivascular injury at both carotid arteries, followed by a 1.5% cholesterol diet. After 90 days, rabbits were randomly divided into 6 groups and treated 5 times with vehicle or ETC-216 at 5, 10, 20, 40, or 150 mg/kg dose every 4 days. Carotid plaque changes were evaluated in vivo by intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI), performed before and at the end of treatments. Magnetic resonance imaging scans were also recorded after administration of the second dose for rabbits infused with vehicle 40 or 150 mg/kg.ResultsAtheroma volume in vehicle-treated rabbits increased dramatically between the first and the second IVUS analyses (+26.53%), whereas in ETC-216–treated animals, a reduced progression at the lower doses and a significant regression at the higher doses, up to −6.83%, was detected. Results obtained by MRI analysis correlated significantly with those at IVUS (r = 0.706; p < 0.0001). The MRI evaluations after the second infusion established that a significant regression was achieved with only 2 administrations of the highest dose.ConclusionsThese results confirm the efficacy of ETC-216 for atherosclerosis treatment and provide guidance for dose selection and frequency to obtain a significant reduction of plaque volume

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.F. B. B. and C. C. acknowledge financial support from the P2RY12 project, ID: GR-2016-02361325 (funded by the Italian Ministry of Health). F. B. B. and P. M. are thankful to the project NiFTy funded by MUR (PRIN2017, no. 2017MYBTXC) and to the NEWMED project, ID: 1175999 (funded by Regione Lombardia POR FESR 2014 2020). The authors acknowledge the Experimental Imaging Center at IRCCS Ospedale San Raffaele, part of the Italian Molecular Imaging node of Euro-BioImaging (https://www.eurobioimaging.eu), for providing access to advanced imaging technologies and services. L. C. and C. C. acknowledge the support of FISM (Fondazione Italiana Sclerosi Multipla onlus) grant no. 2016/R/8. A. M. and G. R. acknowledge the support of Associazione Italiana sulla Ricerca sul Cancro (AIRC: IG 2018, ID. 21763). M. S.-A. thanks the Spanish Government for an FPU PhD Research fellowship and AIRC Foundation for cancer research for a postdoctoral fellowship in Italy. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011 financed by the Instituto de Salud Carlos III with the assistance of the European Regional Development Fund.Peer reviewe

Développements méthodologiques en Imagerie Haute Résolution par Résonance Magnétique Nucléaire (application à la paroi artérielle dans le cas d'athérosclérose)

La visualisation complète de la plaque d'athérosclérose, la détermination de sa composition et de son organisation spatiale, sont nécessaires afin de prévoir sa rupture. L'imagerie par RMN, reconnue pour sa capacité de caractérisation tissulaire, apparaît être une méthode de choix pour l'identification et le suivi des lésions d'athérosclérose. Le travail décrit dans ce manuscrit est consacré aux développements méthodologiques nécessaires pour observer et analyser la plaque d'athérosclérose chez l'homme et sur des modèles animaux. Dans le premier chapitre une description basique est faite de la paroi vasculaire normale et pathologique, et des méthodes d'imagerie de la plaque. Dans le deuxième chapitre, les principes de formation du signal RMN et de l'image RMN sont présentés dans la perspective d'imagerie à haute résolution spatiale pour ensuite dégager les facteurs limitant la résolution et la sensibilité de la mesure. Le troisième chapitre est dédié à des études de la paroi vasculaire, avec dans un premier temps des expériences sur des prélèvements d'artères humaines pour évaluer et analyser les contrastes IRM. Ces résultats sont suivis par une étude sur modèles animaux : à l'échelle microscopique (souris) et à une échelle plus large (lapins). Ce troisième chapitre permet d'exposer les difficultés pour caractériser les composantes de la plaque en raison des limitations en résolution spatiale et en rapport signal sur bruit. De nouvelles approches d'observation par IRM sont nécessaires. Dans cette optique, nous avons employé des agents de contraste IRM qui permettent d'améliorer le contraste tissulaire et de mettre en évidence des propriétés fonctionnelles de la plaque telles que la perméabilité de l'endothélium qui joue un grand rôle dans la formation et l'évolution de la plaque. Dans le dernier chapitre, le développement de d'antennes RMN endoluminales est proposé pour améliorer le rapport signal sur bruit. Cette dernière approche permet une amélioration considérable de l'observation et donc de l'analyse de la paroi artérielle.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

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 vivo19F-MRI, across the whole body, and microscopic imaging at tissue and cellular levels by Raman imaging

Hydrophobin-Coated Solid Fluorinated Nanoparticles for 19F-MRI

Funding Information: N.A., V.D., P.M., and F.B.B. are thankful to the NEWMED project, ID: 1175999 (funded by Regione Lombardia POR FESR 2014 2020). F.B.B. and P.M. are also thankful to the project NiFTy funded by MIUR (PRIN2017, no. 2017MYBTXC). C.C. and F.B.B. are also thankful to the P2RY12 project, ID: GR‐2016‐02361325 (funded by the Italian Ministry of Health). The authors acknowledge the provision of facilities and technical support by Aalto University at OtaNano—Nanomicroscopy Center (Aalto‐NMC). Prof. Pompea Del Vecchio is acknowledged for micro‐DSC measurements on HFBII‐FNP dispersions. Publisher Copyright: © 2021 The Authors. Advanced Materials Interfaces published by Wiley-VCH GmbHIn recent years, fluorine-magnetic resonance imaging (19F-MRI) has emerged as a promising diagnostic technique, complementary to traditional proton magnetic resonance imaging (1H-MRI) and easily translatable for clinical use, providing in-depth in vivo quantification without the use of radioactive agents. This creates a need for the development of appropriate delivery systems for highly omniphobic fluorinated probes. The use of the film-forming protein hydrophobin (HFBII) represents a sustainable and simple method to invert the philicity of fluorinated surfaces. Here, the ability of HFBII to form a rigid protein monolayer on superfluorinated coatings rendering them hydrophilic is shown, a property that is also retained in biological environment. This approach is then translated to directly disperse a solid superfluorinated 19F-MRI probe, PERFECTA, in aqueous solution through the formation of core-shell hydrophobin stabilized PERFECTA nanoparticles (NPs). The obtained NPs are fully characterized in terms of morphology, magnetic properties, colloidal stability, protein corona formation, cellular viability, and imaging performance.Peer reviewe

A comprehensive longitudinal study of magnetic resonance imaging identifies novel features of the Mecp2 deficient mouse brain

Rett syndrome (RTT) is a X-linked neurodevelopmental disorder which represents the leading cause of severe incurable intellectual disability in females worldwide. The vast majority of RTT cases are caused by mutations in the X-linked MECP2 gene, and preclinical studies on RTT largely benefit from the use of mouse models of Mecp2, which present a broad spectrum of symptoms phenocopying those manifested by RTT patients.Neurons represent the core targets of the pathology; however, neuroanatomical abnormalities that regionally characterize the Mecp2 deficient mammalian brain remain ill-defined.Neuroimaging techniques, such as MRI and MRS, represent a key approach for assessing in vivo anatomic and metabolic changes in brain. Being non-invasive, these analyses also permit to investigate how the disease progresses over time through longitudinal studies.To foster the biological comprehension of RTT and identify useful biomarkers, we have performed a thorough in vivo longitudinal study of MRI and MRS in Mecp2 deficient mouse brains. Analyses were performed on both genders of two different mouse models of RTT, using an automatic atlas-based segmentation tool that permitted to obtain a detailed and unbiased description of the whole RTT mouse brain. We found that the most robust alteration of the RTT brain consists in an overall reduction of the brain volume. Accordingly, Mecp2 deficiency generally delays brain growth, eventually leading, in heterozygous older animals, to stagnation and/or contraction. Most but not all brain regions participate in the observed deficiency in brain size; similarly, the volumetric defect progresses diversely in different brain areas also depending on the specific Mecp2 genetic lesion and gender. Interestingly, in some regions volumetric defects anticipate overt symptoms, possibly revealing where the pathology originates and providing a useful biomarker for assessing drug efficacy in pre-clinical studies

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.F.B.B. and P.M. are thankful to the project NiFTy funded by MUR (PRIN2017, no. 2017MYBTXC) and to the NEWMED project, ID: 1175999 (funded by Regione Lombardia POR FESR 2014 2020). F.B.B. and C.C. also acknowledge financial support from P2RY12 project, ID: GR-2016- 02361325 (funded by the Italian Ministry of Health). We would like to thank Dr Marina Cretich from “Istituto di Scienze e Tecnologie Chimiche” (SCITEC-CNR) in Milan for the use of NTA Instrument. Experimental Imaging Center at IRCCS Ospedale San Raffaele is acknowledged for access to the preclinical MRI scanner. M S-A thanks the Spanish Government for an FPU PhD Research fellowship and AIRC- Fundation for cancer research for a Postdoctoral fellowship in Italy. CIBER-BBN (initiative funded by the VI National R&D&i Plan 2008– 2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund) and Nanbiosis ICTS are gratefully acknowledged. J.S. acknowledges the European Research Council (ERC Advanced Grant CADENCE number 742684). The electron microscopy studies were conducted at the Laboratorio de Microscopias Avanzadas-ICTS ELECMI, Universidad de Zaragoza, Spain.Peer reviewe