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)

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

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)

Three-Dimensional Conductive Scaffolds as Neural Prostheses Based on Carbon Nanotubes and Polypyrrole

Unformatted post printThree-dimensional scaffolds for cellular organization need to enjoy a series of specific properties. On the one hand, the morphology, shape and porosity are critical parameters and eventually related with the mechanical properties. On the other hand, electrical conductivity is an important asset when dealing with electroactive cells, so it is a desirable property even if the conductivity values are not particularly high. Here, we construct three-dimensional (3D) porous and conductive composites, where C8-D1A astrocytic cells were incubated to study their biocompatibility. The manufactured scaffolds are composed exclusively of carbon nanotubes (CNTs), a most promising material to interface with neuronal tissue, and polypyrrole (PPy), a conjugated polymer demonstrated to reduce gliosis, improve adaptability, and increase charge-transfer efficiency in brain-machine interfaces. We developed a new and easy strategy, based on the vapor phase polymerization (VPP) technique, where the monomer vapor is polymerized inside a sucrose sacrificial template containing CNT and an oxidizing agent. After removing the sucrose template, a 3D porous scaffold was obtained and its physical, chemical, and electrical properties were evaluated. The obtained scaffold showed very low density, high and homogeneous porosity, electrical conductivity, and Young’s Modulus similar to the in vivo tissue. Its high biocompatibility was demonstrated even after 6 days of incubation, thus paving the way for the development of new conductive 3D scaffolds potentially useful in the field of electroactive tissues.MP received funding from the Spanish Ministry of Economy and Competitiveness MINECO (project CTQ2016-76721-R), Diputación Foral de Gipuzkoa program Red (101/16) and ELKARTEK bmG2017 (Ref: Elkartek KK-2017/00008, BOPV resolution: 8 Feb 2018). NA has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 753293, acronym NanoBEAT. We acknowledge Donato Mancino for the support given during the revision stage. As well, AXA Research Fund and University of Trieste are gratefully acknowledged