Radiomarcaje y estudios de biodistribución de nanopartículas poliméricas como adyuvantes para la vacunación oftálmica frente a la brucelosis

Objetivos: Optimizar el radiomarcaje con 99mTc de nanopartículas de Gantrez® manosiladas y cargadas con el antígeno de Brucella Ovis (Man-NP-HS) y llevar a cabo estudios de biodistribución en ratón tras la administración de las nanopartículas por vía ocular. Metodología: Las Man-NP-HS se obtuvieron por el método de desplazamiento de disolvente. Se purificaron, liofilizaron y caracterizaron. A continuación, se marcaron con 74 MBq de 99mTcO4 - previamente reducido con una disolución ácida de cloruro de estaño, trabajando en ausencia de oxígeno y con un pH final de 4. El rendimiento del marcaje se evaluó mediante TLC. Los estudios de biodistribución se llevaron a cabo en ratones tras la administración oftálmica de la formulación y de un control de 99mTcO4 - libre. Para ello, se sacrificaron los animales a las 2 y a las 24 horas tras la administración ocular y se contaron los órganos en un contador gamma. Resultados: Se obtuvo un rendimiento de marcaje superior al 90%. Los estudios de biodistribución de 99mTc-Man-NP-HS permitieron detectar la actividad concentrada en mucosa nasal y ocular y tracto gastrointestinal tanto a las 2 como a las 24 horas, frente a la biodistribución de 99mTcO4 - libre que permaneció concentrado en la piel alrededor del ojo y en tracto gastrointestinal. Conclusión: Los estudios de biodistribución de 99mTc-Man-NP-HS tras administración oftálmica han permitido demostrar su biodistribución en mucosas y tracto gastrointestinal, característica indispensable como sistema de liberación de antígenos a través de mucosa ocular. Esto, junto con su elevada respuesta inmune, efectiva protección y no virulencia, convierte a estas nanopartículas en una vacuna ideal anti Brucelosis

Zein nanoparticles for oral folic acid delivery

The aim of this work was to prepare and evaluate the capability of zein nanoparticles for oral drug delivery. More particularly, in this work, the ability of these nanoparticles to improve the oral bioavailability of folic acid is reported. The nanoparticles were prepared by a desolvation process, followed by purification via ultrafiltration and drying in a spray-drier apparatus. The resulting nanoparticles displayed a mean size close to 200 nm with negative zeta potential and a payload of 54 μg folic acid per mg nanoparticle. From the in vitro release studies, it was observed that folic acid was only released from nanoparticles in simulated intestinal conditions. In vivo biodistribution studies,with radiolabelled or fluorescently marked nanoparticles, revealed that nanoparticles remained within the gut and were capable of interacting with the protective mucus layer of the jejunum. For the pharmacokinetic study, folic acid was orally administered to rats as a single dose of 1 mg/kg.The relatively oral bioavailability of folic acid, when encapsulated in zein nanoparticles, was around 70%: two-times higher than the value obtained with an aqueous solution of the vitamin. This fact might be explained by the mucoadhesive properties of these nanoparticles

Tomografía por emisión de positrones en el cáncer de mama

PET18FDG is an imaging diagnostic technique that shows changes in glycolitic metabolism that appear at a very early phases in the tumoral process. The main limitation of PET in breast cancer is the detection of small tumor lesions and axillary micrometastases. However it offers important information in the staging of high risk patients, in clinical relapse or in therapeutic evaluation. The new PET-CT devices offer advantages over conventional techniques. It provides a greater precision in the localization of tumoral foci. In spite of current difficulties for clinical applications, fluoro-estradiol (18F-ES) offers the possibilty of studying the presence of estrogenic receptors both in the primary and in the metastases. It may prove to be a useful tool to obtain information about therapeutic management and prognosis of breast cancer

Evaluation of spacial resolution of a PET scanner through the simulation and experimental measurement of the Recovery coefficient

Purpose: In order to measure spatial resolution of a PET tomograph in clinical conditions, this study describes and validates a method based on the recovery coefficient, a factor required to compensate underestimation in measured radioactivity concentration for small structures. Methods: In a PET image, the recovery factors of radioactive spheres were measured and their comparison with simulated recovery coefficients yielded the tomographic spatial resolution. Following this methodology, resolution was determined in different surrounding media and several conditions for reconstruction, including clinical conditions for brain PET studies. All spatial resolution values were compared with those obtained using classical methods with point and line sources. Results: In each considered condition, spatial resolution of the PET image estimated using the recovery coefficient showed good agreement with classical methods measurements, validating the procedure. Conclusion: Measurement of the recovery coefficient provides an assessment of tomographic spatial resolution, particularly in clinical studies conditions

Dissolving microneedles for intradermal vaccination against shigellosis

Intradermal (ID) immunization is of increasing interest due to the easy accessibility and excellent immunogenic properties of the skin. Among ID immunization methods, dissolving microneedles (MNs) have appeared as an alternative to traditional hypodermic immunization, offering many advantages, such as being an easily administered method, with no need for health personnel, painless, and avoiding the use of needles and sharp wastage. In this study, an affordable and easy-to-produce MNs method was developed based on aqueous blends of 30% w/w poly (methyl vinyl ether-co-maleic anhydride). As an antigen model, a subunit vaccine candidate based on outer membrane vesicles from Shigella flexneri was used. Both unloaded and antigen-loaded MNs were synthetized and characterized. The MNs were successfully validated in an in vitro Parafilm M skin model and in a pig skin ex vivo model. Biodistribution studies were performed in BALB/c mice using 99mTcO4- radiolabeled samples. Results indicated that the vesicle vaccine was successfully released from the MNs and targeted gastrointestinal tract after 6 h post-administration. In vivo immunization and protection studies were performed in BALB/c mice. Mice were intradermally immunized through ear skin with one single dose of 200 g antigenic complex, eliciting the production of specific systemic IgG and mucosal IgA

New MRI, 18F-DOPA and 11C-(+)-alpha-dihydrotetrabenazine templates for Macaca fascicularis neuroimaging: advantages to improve PET quantification

Normalization of neuroimaging studies to a stereotaxic space allows the utilization of standard volumes of interest (VOIs) and voxel-based analysis (SPM). Such spatial normalization of PET and MRI studies requires a high quality template image. The aim of this study was to create new MRI and PET templates of 18F-DOPA and 11C-(+)-α-dihydrotetrabenazine (11C-DTBZ) of the Macaca fascicularis brain, an important animal model of Parkinson's disease. MRI template was constructed as a smoothed average of the scans of 15 healthy animals, previously transformed into the space of one representative MRI. In order to create the PET templates, 18F-DOPA and 11C-DTBZ PET of the same subjects were acquired in a dedicated small animal PET scanner and transformed to the created MRI template space. To validate these templates for PET quantification, parametric values obtained with a standard VOI-map applied after spatial normalization to each template were statistically compared to results computed using individual VOIs drawn for each animal. The high correlation between both procedures validated the utilization of all the templates, improving the reproducibility of PET analysis. To prove the utility of the templates for voxel-based quantification, dopamine striatal depletion in a representative monkey treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was assessed by SPM analysis of 11C-DTBZ PET. A symmetric reduction in striatal 11C-DTBZ uptake was detected in accordance with the induced lesion. In conclusion, templates of M. fascicularis brain have been constructed and validated for reproducible and automated PET quantification. All templates are electronically available via the internet

Desarrollo de procedimientos automatizados de síntesis de radiofármacos PET marcados con carbono-11 a temperatura ambiente y purificación mediante extracción en fase sólida

The continued increase in the number of PET studies with carbon-11 labelled radiopharmaceuticals, along with the limitations in space and laboratory equipment is a real problem and a radiation protection challenge. Adaptation of synthesis procedures to solid-phase supported approaches implies a global simplification of the synthesis system, an increase in safety and a substantial decrease in the overall necessary time to carry out the synthesis. Objectives The aims of this study were: (1) to design, assemble and test a fully-automated solid-phase supported synthesis system based on commercial modules for the simultaneous production of L-[Methyl-(11C)]Methionine (MET) and [11C]-Choline (CHOL); (2) set-up such system also for the routine production of [11C]-(+)-á- dihydrotetrabenazine (DTBZ); and (3) design and test a solid phase extraction based simplified system using whole-sterile single use "plug & play" kits for synthesis of MET and CHOL using Eckert&Ziegler Modular Lab modules. Methodology A commercial Tracerlab FXc module (GE) was modified to carry out the simultaneous production of MET and CHOL starting from [11C]methyl iodide. A completely automated synthesis sequence was programmed and 48 synthesis runs made to demonstrate performance of the system. Reaction times, synthesis yields and full QC of both radiopharmaceuticals were carried out. Such system was further adapted (including control interface and running sequences) for the synthesis of DTBZ using two Alumina N cartridges for final purification of the product and 54 synthesis runs carried out. Solvents were then evaporated under vacuum with helium flow. Finally, for the "plug and play" approach kits were set-up in a LFC using single-use sterile medical material, sterilised and further used for the synthesis of MET and CHOL using the designed Modular Lab based synthesis system. A control interface and a synthesis sequence programs were also designed for such purpose. Results and conclusions Simultaneous production of MET and CHOL was finalised in just six minutes with mean productions of 2.4 and 2.7 GBq respectively, and radiopharmaceutical purity according to pharmacopoeia standards. [11C]DTBZ (1.94 ±0.13 GBq) was obtained with radiochemical purity >99% while residual solvents were below PhEur stablished levels. The ¿plug &play¿ system produced MET and CHOL with radiopharmaceutical purity with 5.0 and 5.3 GBq mean production yields. The new procedures minimises the time required for the preparation of the system, avoids cleaning runs, facilitates fulfilment of GMP quality standards and improves radiation protection to personnel

Desarrollo de procedimientos automatizados de síntesis de radiofármacos PET marcados con carbono-11 a temperatura ambiente y purificación mediante extracción en fase sólida

The continued increase in the number of PET studies with carbon-11 labelled radiopharmaceuticals, along with the limitations in space and laboratory equipment is a real problem and a radiation protection challenge. Adaptation of synthesis procedures to solid-phase supported approaches implies a global simplification of the synthesis system, an increase in safety and a substantial decrease in the overall necessary time to carry out the synthesis. Objectives The aims of this study were: (1) to design, assemble and test a fully-automated solid-phase supported synthesis system based on commercial modules for the simultaneous production of L-[Methyl-(11C)]Methionine (MET) and [11C]-Choline (CHOL); (2) set-up such system also for the routine production of [11C]-(+)-á- dihydrotetrabenazine (DTBZ); and (3) design and test a solid phase extraction based simplified system using whole-sterile single use "plug & play" kits for synthesis of MET and CHOL using Eckert&Ziegler Modular Lab modules. Methodology A commercial Tracerlab FXc module (GE) was modified to carry out the simultaneous production of MET and CHOL starting from [11C]methyl iodide. A completely automated synthesis sequence was programmed and 48 synthesis runs made to demonstrate performance of the system. Reaction times, synthesis yields and full QC of both radiopharmaceuticals were carried out. Such system was further adapted (including control interface and running sequences) for the synthesis of DTBZ using two Alumina N cartridges for final purification of the product and 54 synthesis runs carried out. Solvents were then evaporated under vacuum with helium flow. Finally, for the "plug and play" approach kits were set-up in a LFC using single-use sterile medical material, sterilised and further used for the synthesis of MET and CHOL using the designed Modular Lab based synthesis system. A control interface and a synthesis sequence programs were also designed for such purpose. Results and conclusions Simultaneous production of MET and CHOL was finalised in just six minutes with mean productions of 2.4 and 2.7 GBq respectively, and radiopharmaceutical purity according to pharmacopoeia standards. [11C]DTBZ (1.94 ±0.13 GBq) was obtained with radiochemical purity >99% while residual solvents were below PhEur stablished levels. The ¿plug &play¿ system produced MET and CHOL with radiopharmaceutical purity with 5.0 and 5.3 GBq mean production yields. The new procedures minimises the time required for the preparation of the system, avoids cleaning runs, facilitates fulfilment of GMP quality standards and improves radiation protection to personnel

Radiomarcaje y biodistribución mediante SPECT/CT de nanopartículas de bevacizumab

Introducción: la nanomedicina avanza en el diseño de nanopartículas para transporte y liberación de fármacos. Un mismo nanotransportador puede combinar agentes terapéuticos y de imagen. El radiomarcaje con radionúclidos es uno de los procedimientos más utilizados para ello. Existen diversas estrategias para realizar radiomarcajes de NPs. Casi todas ellas se centran en el radiomarcaje del vehículo, asumiendo que la liberación de la carga se produce donde se visualiza el foco de captación y obviando el destino final real del mismo. Una evaluación más exacta sería el radiomarcaje de la carga que permite visualizarlo encapsulado y tras su liberación. Objetivo: ha sido el radiomarcaje con tecnecio-99m de distintas formulaciones de NP-HSA, vacías o cargadas bevacizumab, y la determinación de su biodistribución in vivo mediante imagen SPECT/CT. Metodología y Resultados: se evaluaron las formulaciones NP-HSA-GLU, NP-HSA-PEG y NP-HSA-Gantrez como posible vehículo de liberación de bevacizumab. Se radiomarcaron superficialmente con tecnecio-99m empleando un sistema tipo kit y se valoró su distribución mediante SPECT/CT en modelo animal de rata Wistar sana tras administración oftálmica. Las imágenes evidenciaron que todas permanecieron 10h en el lugar de la administración con mayor actividad en las NP-HSA-PEG y NP-HSA-GLU. Estas se emplearon para encapsular el bevacizumab (NP-HSA-B-PEG y NP-HSA-B-GLU), cuya distribución se valoró comparativamente mediante SPECT/CT con las nanopartículas desnudas (NP-HSA-B), mediante radiomarcaje de cada tipo con tecnecio-99m. De cada formulación se valoraron los parámetros fisicoquímicos antes y después del radiomarcaje, y su distribución de igual modo que las vacías. Los resultados indicaron que las formulaciones mantuvieron las características fisicoquímicas tras su radiomarcaje. Todas permanecieron en el ojo 8h, pero la excreción de las NP-HSA-B fue la más rápida y el porcentaje de permanencia en superficie ocular (%PSR) fue menor a las formulaciones recubiertas. Por el contrario, el %PRS de las NP-HSA-B-PEG fue 2veces superior hasta las 6h, considerándose que reunía las mejores características. Se radiomarcó con tecnecio-99m el bevacizumab mediante 2métodos diferentes, de loa cuales el más adecuado fue el radiomarcaje con [99mTc]Tc(CO)3(H2O)3. El [99mTc]Tc(CO)3-Bevacizumab se encapsuló en una matriz HSA-PEG. Para valorar el destino final de las diferentes partes del nanosistema NP-HSA-B-PEG(cubierta y agente terapéutico) se realizaron estudios de biodistribución in vivo mediante SPECT/CT tras administración intravenosa de ambas formulaciones.Los resultados pusieron de manifiesto que las nanopartículas mostraron una menor captación inespecífica que los controles, sin captación estomacal, esplénica o tiroidea. La distribución de las formulaciones [99mTc]Tc‐NP‐HSA‐PEG y [99mTc]Tc‐NP‐HSA‐B‐PEG fue similar; evidenciando una elevada captación hepática y renal. Tras 4h la captación renal del NP‐HSA‐[99mTc]Tc(CO)3-Bevacizumab-PEG fue similar que las formulaciones radiomarcadas externamente tras 24h. Conclusiones: el sistema tipo kit es un método adecuado radiomarcar nanopartículas. Los estudios de biodistribución indicaron que las formulaciones permanecieron en ojo durante 8h tras su administración, influyendo la modificación superficial en ello. La eliminación de las formulaciones fué principalmente gastrointestinal y más lenta en aquellas cargadas con bevacizumab. El estudio comparativo de biodistribución, en función del lugar del radiomarcaje, mostró diferencias entre los perfiles de eliminación de las mismas, a pesar de ser el mismo nanosistema

Radiomarcaje de nanopartículas de albúmina con radiometales (Tecnecio-99M, Galoo-67, Zirconio-89) y estudio de su biodistribución mediante imagen molecular

Las nanopartículas de albúmina pueden utilizarse para la encapsulación de fármacos, permitiendo una liberación controlada y dirigida de los mismos. Además, su superficie puede modificarse mediante el recubrimiento con polímeros, mejorando sus propiedades físico-químicas. Sin embargo, resulta extremadamente difícil conocer el comportamiento in vivo de estas nanopartículas tras su administración, lo cual es esencial a la hora de determinar su efectividad o sus posibles efectos secundarios. El radiomarcaje de nanopartículas con isótopos emisores gamma o emisores de positrones y la adquisición de imágenes mediante técnicas de imagen molecular, puede proporcionar información relevante sobre el comportamiento de estas nanopartículas in vivo. En esta tesis doctoral, se han marcado radiactivamente con tecnecio-99m, galio-67 y zirconio-89 nanopartículas de albúmina recubiertas con los polímeros hidroxipropilmetilcelulosa (HPMC) y los polímeros conjugados de Gantrez-AN con la tiamina (GTM2), manosamina (GMN2) y polietilenglicol 2000 (GPM2). Las nanopartículas se prepararon empleando el método de desolvatación. Posteriormente las nanopartículas se marcaron mediante marcaje directo con pertecnetato sódico de tecnecio-99 y con cloruro de galio-67 mediante la formación de un complejo quelante-radiometal, empleando el quelante NOTA. Tras estudiar la estabilidad in vitro del marcaje en suero fisiológico 0,9% y plasma sanguíneo, las nanopartículas radiomarcadas se ensayaron in vivo tras su administración intravenosa en ratas Wistar mediante tomografía computerizada por emisión de fotón único (SPECT/CT). Finalmente los animales fueron sacrificados, se extrajeron los órganos y se cuantificó la radiactividad presente en los mismos empleando un contador gamma. Los resultados obtenidos muestran que las nanopartículas de albúmina recubiertas con diferentes polímeros pueden marcarse de forma efectiva con radiometales, obteniéndose elevados rendimientos de marcaje. Las nanopartículas radiomarcadas mostraron una elevada estabilidad in vitro pasadas las 48 horas del marcaje tanto en suero fisiológico como en plasma sanguíneo. Los estudios in vivo permitieron conocer la biodistribución de las nanopartículas durante periodos prolongados de tiempo y los resultados obtenidos se confirmaron con los hallazgos de estudios ex vivo