Radiolabelling and preclinical evaluation of nanoparticles as drug delivery systems: application to infectious pulmonary diseases.

100 p.El trabajo llevado a cabo en esta tesis doctoral se ha dividido en tres partes, que corresponden a los capítulos 3,4 y 5 de la memoria. En la primera parte del trabajo, se presenta el desarrollo de una nueva herramienta para la evaluación de la ventilación pulmonar en ratas sanas (wild type), utilizando la tecnología PET (Tomografía por Emisión de Positrones, de sus siglas en inglés). La herramienta para la determinación de la ventilación pulmonar se basa en la utilización de dos gases radiofluorados, siguiendo una metodología completamente innovadora. En la segunda parte del trabajo, se describe la evaluación y comparación de tres dispositivos diferentes de administración pulmonar de aerosoles en ratas. Para ello, se marcó el aerosol con 2-deoxi-2-(18F)fluoro-D-glucosa ([18F]FDG), un radiotrazador ampliamente conocido en el ámbito clínico principalmente para el diagnóstico precoz y la evaluación de respuesta a tratamientos de diferentes tipos de cáncer, y se efectuaron estudios PET tras administración utilizando los diferentes dispositivos. La tercera y última parte del trabajo ha consistido en la evaluación mediante PET del tiempo de residencia en el pulmón de dos péptidos antimicrobianos tras la administración pulmonar, bien en su forma libre o bien conjugados con diferentes nanovehículos. En primer lugar, se estudiaron diferentes estrategias para incorporar un radioisótopo emisor de positrones o de fotones gamma a los diferentes compuestos (péptidos y nanovehículos). Una vez implementadas las estrategias de marcaje, tanto los péptidos como los nanovehículos como sus combinaciones fueron administrados en ratas wild type y se llevaron a cabo estudios de imagen para determinar el tiempo de residencia en los pulmones tanto de los péptidos marcados como de los nanovehículos seleccionados

Synthesis of Chromen[4,3-b]pyrrolidines by Intramolecular 1,3-Dipolar Cycloadditions of Azomethine Ylides: An Experimental and Computational Assessment of the Origin of Stereocontrol

Azomethine ylides, generated from imine-derived O-cinnamyl or O-crotonyl salicylaldeyde and α-amino acids, undergo intramolecular 1,3-dipolar cycloaddition, leading to chromene[4,3-b]pyrrolidines. Two reaction conditions are used: (a) microwave-assisted heating (200 W, 185 °C) of a neat mixture of reagents, and (b) conventional heating (170 °C) in PEG-400 as solvent. In both cases, a mixture of two epimers at the α-position of the nitrogen atom in the pyrrolidine nucleus was formed through the less energetic endo-approach (B/C ring fusion). In many cases, the formation of the stereoisomer bearing a trans-arrangement into the B/C ring fusion was observed in high proportions. Comprehensive computational and kinetic simulation studies are detailed. An analysis of the stability of transient 1,3-dipoles, followed by an assessment of the intramolecular pathways and kinetics are also reported.Financial support was provided by the Brasilian Universiade Federal do Rio de Janeiro (UFRJ), the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), by the Spanish Ministerio de Ciencia e Innovación (MICINN) (projects CTQ2010-20387, Consolider Ingenio 2010, CSD2007-00006), the Spanish Ministerio de Economía y Competitividad (MINECO) (projects CTQ2013-43446-P, CTQ2014-51912-REDC, and CTQ2013-45415-P), the Fondos Europeos para el Desarrollo Regional (FEDER), the Generalitat Valenciana (PROMETEO 2009/039 and PROMETEOII/2014/017), the Basque Government (GV/EJ, grant IT-324-07), and the Universities of Alicante and of the Basque Country (UPV/EHU) (UFI11/22 QOSYC)

Medical imaging for the tracking of micromotors

Micro/nanomotors are useful tools for several biomedical applications, including targeted drug delivery and minimally invasive microsurgeries. However, major challenges such as in vivo imaging need to be addressed before they can be safely applied on a living body. Here, we show that positron emission tomography (PET), a molecular imaging technique widely used in medical imaging, can also be used to track a large population of tubular Au/PEDOT/Pt micromotors. Chemisorption of an iodine isotope onto the micromotor’s Au surface rendered them detectable by PET, and we could track their movements in a tubular phantom over time frames of up to 15 min. In a second set of experiments, micromotors and the bubbles released during self-propulsion were optically tracked by video imaging and bright-field microscopy. The results from direct optical tracking agreed with those from PET tracking, demonstrating that PET is a suitable technique for the imaging of large populations of active micromotors in opaque environments, thus opening opportunities for the use of this mature imaging technology for the in vivo localization of artificial swimmers

Gold Nanoparticles as Boron Carriers for Boron Neutron Capture Therapy: Synthesis, Radiolabelling and In Vivo Evaluation

Background: Boron Neutron Capture Therapy (BNCT) is a binary approach to cancer therapy that requires accumulation of boron atoms preferentially in tumour cells. This can be achieved by using nanoparticles as boron carriers and taking advantage of the enhanced permeability and retention (EPR) effect. Here, we present the preparation and characterization of size and shape-tuned gold NPs (AuNPs) stabilised with polyethylene glycol (PEG) and functionalized with the boron-rich anion cobalt bis(dicarbollide), commonly known as COSAN. The resulting NPs were radiolabelled with 124I both at the core and the shell, and were evaluated in vivo in a mouse model of human fibrosarcoma (HT1080 cells) using positron emission tomography (PET). Methods: The thiolated COSAN derivatives for subsequent attachment to the gold surface were synthesized by reaction of COSAN with tetrahydropyran (THP) followed by ring opening using potassium thioacetate (KSAc). Iodination on one of the boron atoms of the cluster was also carried out to enable subsequent radiolabelling of the boron cage. AuNPs grafted with mPEG-SH (5 Kda) and thiolated COSAN were prepared by ligand displacement. Radiolabelling was carried out both at the shell (isotopic exchange) and at the core (anionic absorption) of the NPs using 124I to enable PET imaging. Results: Stable gold nanoparticles simultaneously functionalised with PEG and COSAN (PEG-AuNPs@[4]) with hydrodynamic diameter of 37.8 0.5 nm, core diameter of 19.2 1.4 nm and -potential of 18.0 0.7 mV were obtained. The presence of the COSAN on the surface of the NPs was confirmed by Raman Spectroscopy and UV-Vis spectrophotometry. PEG-AuNPs@[4] could be efficiently labelled with 124I both at the core and the shell. Biodistribution studies in a xenograft mouse model of human fibrosarcoma showed major accumulation in liver, lungs and spleen, and poor accumulation in the tumour. The dual labelling approach confirmed the in vivo stability of the PEG-AuNPs@[4]. Conclusions: PEG stabilized, COSAN-functionalised AuNPs could be synthesized, radiolabelled and evaluated in vivo using PET. The low tumour accumulation in the animal model assayed points to the need of tuning the size and geometry of the gold core for future studies

Oral Treatment with Iododiflunisal Delays Hippocampal Amyloid-β Formation in a Transgenic Mouse Model of Alzheimer's Disease: A Longitudinal in vivo Molecular Imaging Study

Transthyretin (TTR) is a tetrameric, amyloid-β (Aβ)-binding protein, which reduces Aβ toxicity. The TTR/Aβ interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer's disease. Objective: We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aβ interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer's disease using positron emission tomography (PET). Methods: Female mice (AβPPswe/PS1A246E/TTR+/-) were divided into 3 groups (n=7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aβ in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[18F]florbetaben. Immunohistochemical analysis was performed at age=14 months. Results: Standard uptake values relative to the cerebellum (SUVr) of [18F]florbetaben in CTX and HIP of non-treated animals progressively increased from age=5 to 11 months and stabilized afterwards. In contrast, [18F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages=5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age=14 months. Conclusion: Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aβ deposition in certain brain regions

In vivo PET Imaging of Gliogenesis After Cerebral Ischemia in Rats

In vivopositron emission tomography of neuroinflammation has mainly focused on the evaluation of glial cell activation using radiolabeled ligands. However, the non-invasive imaging of neuroinflammatory cell proliferation has been scarcely evaluated so far.In vivoandex vivoassessment of gliogenesis after transient middle cerebral artery occlusion (MCAO) in rats was carried out using PET imaging with the marker of cell proliferation 3 '-Deoxy-3 '-[18F] fluorothymidine ([F-18]FLT), magnetic resonance imaging (MRI) and fluorescence immunohistochemistry. MRI-T2W studies showed the presence of the brain infarction at 24 h after MCAO affecting cerebral cortex and striatum.In vivoPET imaging showed a significant increase in [F-18]FLT uptake in the ischemic territory at day 7 followed by a progressive decline from day 14 to day 28 after ischemia onset. In addition, immunohistochemistry studies using Ki67, CD11b, and GFAP to evaluate proliferation of microglia and astrocytes confirmed the PET findings showing the increase of glial proliferation at day 7 after ischemia followed by decrease later on. Hence, these results show that [F-18]FLT provides accurate quantitative information on the time course of glial proliferation in experimental stroke. Finally, this novel brain imaging method might guide on the imaging evaluation of the role of gliogenesis after stroke.The authors would like to thank A. Leukona, X. Rios-Anglada, and V. Salinas for technical support in the radiosynthesis. This study was funded by grants from the Spanish Ministry of Education and Science/FEDER RYC-2017-22412, SAF2016-75292-R, PID2019-107989RB-I00, the Basque Government (IT1203/19, BIO18/IC/006) and CIBERNED. Maria Ardaya holds a fellowship from the University of Pais Vasco. Ana Joya acknowledges funding from Fundacio La Marato de TV3 (17/C/2017). Part of the work has been performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM-2017-0720)

Longitudinal evaluation of neuroinflammation and oxidative stress in a mouse model of Alzheimer disease using positron emission tomography

[EN] Background: Validation of new biomarkers of Alzheimer disease (AD) is crucial for the successful development and implementation of treatment strategies. Additional to traditional AT(N) biomarkers, neuroinflammation biomarkers, such as translocator protein (TSPO) and cystine/glutamine antiporter system (x(c)(-)), could be considered when assessing AD progression. Herein, we report the longitudinal investigation of [F-18]DPA-714 and [F-18]FSPG for their ability to detect TSPO and x(c)(-) biomarkers, respectively, in the 5xFAD mouse model for AD. Methods: Expression of TSPO and x(c)(-) system was assessed longitudinally (2-12 months of age) on 5xFAD mice and their respective controls by positron emission tomography (PET) imaging using radioligands [F-18]DPA-714 and [F-18]FSPG. In parallel, in the same mice, amyloid-beta plaque deposition was assessed with the amyloid PET radiotracer [F-18]florbetaben. In vivo findings were correlated to ex vivo immunofluorescence staining of TSPO and x(c)(-) in microglia/macrophages and astrocytes on brain slices. Physiological changes of the brain tissue were assessed by magnetic resonance imaging (MRI) in 12-month-old mice. Results: PET studies showed a significant increase in the uptake of [F-18]DPA-714 and [F-18]FSPG in the cortex, hippocampus, and thalamus in 5xFAD but not in WT mice over time. The results correlate with A beta plaque deposition. Ex vivo staining confirmed higher TSPO overexpression in both, microglia/macrophages and astrocytes, and overexpression of x(c)(-) in non-glial cells of 5xFAD mice. Additionally, the results show that A beta plaques were surrounded by microglia/macrophages overexpressing TSPO. MRI studies showed significant tissue shrinkage and microstructural alterations in 5xFAD mice compared to controls. Conclusions: TSPO and x(c)(-) overexpression can be assessed by [F-18]DPA-714 and [F-18]FSPG, respectively, and correlate with the level of A beta plaque deposition obtained with a PET amyloid tracer. These results position the two tracers as promising imaging tools for the evaluation of disease progression.J.L. and P.R. thank the Spanish Ministry of Science and Innovation MCIN/AEI/10.13039/501100011033 (PID2020-117656RB-100 and PID2020-118546RBI00, respectively) and the Interreg Atlantic Area Programme (EAPA_791/2018). Abraham Martin acknowledges funding from the Spanish Ministry of Education and Science (RYC-2017-22412, PID2019-107989RB-I00), the Basque Government (BIO18/IC/006), and Fundacio La Marato de TV3 (17/C/2017). Estibaliz Capetillo-Zarate acknowledges funding from the Basque Government (IT120319; ELKARTEK KK-2020/00034) and CIBERNED (CB06/0005/0076). The work was performed under the Maria de Maeztu Units of Excellence Programme -Grant MDM-2017-0720 funded by MCIN/AEI/10.13039/50110001103

Longitudinal evaluation of a novel BChE PET tracer as an early in vivo biomarker in the brain of a mouse model for Alzheimer disease

Purpose: The increase in butyrylcholinesterase (BChE) activity in the brain of Alzheimer disease (AD) patients and animal models of AD position this enzyme as a potential biomarker of the disease. However, the information on the ability of BChE to serve as AD biomarker is contradicting, also due to scarce longitudinal studies of BChE activity abundance. Here, we report 11C-labeling, in vivo stability, biodistribution, and longitudinal study on BChE abundance in the brains of control and 5xFAD (AD model) animals, using a potent BChE selective inhibitor, [11C]4, and positron emission tomography (PET) in combination with computerised tomography (CT). We correlate the results with in vivo amyloid beta (Aβ) deposition, longitudinally assessed by [18F]florbetaben-PET imaging. Methods: [11C]4 was radiolabelled through 11C-methylation. Metabolism studies were performed on blood and brain samples of female wild type (WT) mice. Biodistribution studies were performed in female WT mice using dynamic PET-CT imaging. Specific binding was demonstrated by ex vivo and in vivo PET imaging blocking studies in female WT and 5xFAD mice at the age of 7 months. Longitudinal PET imaging of BChE was conducted in female 5xFAD mice at 4, 6, 8, 10 and 12 months of age and compared to age-matched control animals. Additionally, Aβ plaque distribution was assessed in the same mice using [18F]florbetaben at the ages of 2, 5, 7 and 11 months. The results were validated by ex vivo staining of BChE at 4, 8, and 12 months and Aβ at 12 months on brain samples. Results: [11C]4 was produced in sufficient radiochemical yield and molar activity for the use in PET imaging. Metabolism and biodistribution studies confirmed sufficient stability in vivo, the ability of [11C]4 to cross the blood brain barrier (BBB) and rapid washout from the brain. Blocking studies confirmed specificity of the binding. Longitudinal PET studies showed increased levels of BChE in the cerebral cortex, hippocampus, striatum, thalamus, cerebellum and brain stem in aged AD mice compared to WT littermates. [18F]Florbetaben-PET imaging showed similar trend of Aβ plaques accumulation in the cerebral cortex and the hippocampus of AD animals as the one observed for BChE at ages 4 to 8 months. Contrarily to the results obtained by ex vivo staining, lower abundance of BChE was observed in vivo at 10 and 12 months than at 8 months of age. Conclusions: The BChE inhibitor [11C]4 crosses the BBB and is quickly washed out of the brain of WT mice. Comparison between AD and WT mice shows accumulation of the radiotracer in the AD-affected areas of the brain over time during the early disease progression. The results correspond well with Aβ accumulation, suggesting that BChE is a promising early biomarker for incipient AD

In vivo multimodal imaging of adenosine A1 receptors in neuroinflammation after experimental stroke

Adenosine A(l) receptors (A(l)ARs) are promising imaging biomarkers and targets for the treatment of stroke. Nevertheless, the role of A(l)ARs on ischemic damage and its subsequent neuroinflammatory response has been scarcely explored so far. Methods: In this study, the expression of A(1)ARs after transient middle cerebral artery occlusion (MCAO) was evaluated by positron emission tomography (PET) with [F-18]CPFPX and immunohistochemistry (IHC). In addition, the role of AIARs on stroke inflammation using pharmacological modulation was assessed with magnetic resonance imaging (MRI), PET imaging with [F-18]DPA-714 (TSPO) and [F-18]FLT (cellular proliferation), as well as IHC and neurofunctional studies. Results: In the ischemic territory, [F-18]CPFPX signal and IHC showed the overexpression of A(l)ARs in microglia and infiltrated leukocytes after cerebral ischemia. Ischemic rats treated with the AAR agonist ENBA showed a significant decrease in both [F-18]DPA-714 and [F-18]FLT signal intensities at day 7 after cerebral ischemia, a feature that was confirmed by IHC results. Besides, the activation of A(l)AR promoted the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome including motor, sensory and reflex responses. These results show for the first time the in vivo PET imaging of A(l)AR expression after cerebral ischemia in rats and the application of [F-18]FLT to evaluate glial proliferation in response to treatment. Conclusion: Notably, these data provide evidence for A(l)AR playing a key role in the control of both the activation of resident glia and the de novo proliferation of microglia and macrophages after experimental stroke in rats.The authors would like to thank A. Leukona and V. Salinas for technical support in the radiosynthesis. This study was funded by grants from the Spanish Ministry of Education and Science/FEDER RYC-201722412, SAF2016-75292-R, SAF2017-87670-R and PID2019-107989RB-I00, the Basque Government (IT1203/19, BIO18/IC/006) and CIBERNED. Maria Ardaya holds a fellowship from the University of Pais Vasco. Ana Joya acknowledges funding from Fundacio La Marato de TV3 (17/C/2017). Juan Jose Gutierrez acknowledges funding from Euskampus Fundazioa. Jordi Llop also acknowledges The Spanish Ministry of Economy and Competitiveness (Grant CTQ2017-87637-R). Part of the work has been performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM-2017-0720)

Radiochemical examination of transthyretin (TTR) brain penetration assisted by iododiflunisal, a TTR tetramer stabilizer and a new candidate drug for AD

It is well settled that the amyloidogenic properties of the plasma protein transporter transthyretin (TTR) can be modulated by compounds that stabilize its native tetrameric conformation. TTR is also present in cerebrospinal fluid where it can bind to Aβ-peptides and prevent Aβ aggregation. We have previously shown that treatment of Alzheimer’s Disease (AD) model mice with iododiflunisal (IDIF), a TTR tetramer stabilizing compound, prevents AD pathologies. This evidence positioned IDIF as a new lead drug for AD. In dissecting the mechanism of action of IDIF, we disclose here different labeling strategies for the preparation of 131I-labeled IDIF and 131I- and 124I-labeled TTR, which have been further used for the preparation of IDIF-TTR complexes labeled either on the compound or the protein. The biodistribution of all labeled species after intravenous administration has been investigated in mice using ex vivo and in vivo techniques. Our results confirm the capacity of TTR to cross the blood brain barrier (BBB) and suggest that the formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR. The increased TTR and IDIF brain concentrations may result in higher Aβ-peptide sequestration capacity with the subsequent inhibition of AD symptoms as we have previously observed in mice. © 2019, The Author(s).The work was supported by a grant from the Fundació Marató de TV3 (Neurodegenerative Diseases Call, Project Reference 20140330-31-32-33-34, http://www.ccma.cat/tv3/marato/en/ projectes-financats/2013/212/). The group at CIC biomaGUNE also acknowledges MINECO (Spain) for funding through Grant CTQ2017-87637-R. I. Cardoso worked under the Investigator FCT Program which is financed by national funds through the Foundation for Science and Technology (FCT, Portugal) and co-financed by the European Social Fund (ESF) through the Human Potential Operational Programme (HPOP), type 4.2 - Promotion of Scientific Employment.Peer reviewe