A New Porphyrin for the Preparation of Functionalized Water-Soluble Gold Nanoparticles with Low Intrinsic Toxicity

A potential new photosensitizer based on a dissymmetric porphyrin derivative bearing a thiol group was synthesized. 5-[4-(11-Mercaptoundecyloxy)-phenyl-10,15,20-triphenylporphyrin (PR-SH) was used to functionalize gold nanoparticles in order to obtain a potential drug delivery system. Water-soluble multifunctional gold nanoparticles GNP-PR/PEG were prepared using the Brust-Schiffrin methodology, by immobilization of both a thiolated polyethylene glycol (PEG) and the porphyrin thiol compound (PR-SH). The nanoparticles were fully characterized by transmission electron microscopy and 1H nuclear magnetic resonance spectroscopy, UV/Vis absorption spectroscopy, and X-ray photoelectron spectroscopy. Furthermore, the ability of GNP-PR/PEGs to induce singlet oxygen production was analyzed to demonstrate the activity of the photosensitizer. Cytotoxicity experiments showed the nanoparticles are nontoxic. Finally, cellular uptake experiments demonstrated that the functionalized gold nanoparticles are internalized. Therefore, this colloid can be considered to be a novel nanosystem that could potentially be suitable as an intracellular drug delivery system of photosensitizers for photodynamic therapy. Keywords: drug delivery, gold nanoparticles, photodynamic therapy, photosensitizers, porphyrin

Synthesis and functionalization of nano- and micro-particles for sensing and therapy in living cells

[spa] El diseño y la preparación, mediante la utilización de procesos de biofuncionalización de micro / nanosistemas que puedan tener aplicación en células vivas es un tema de actualidad en campos como la Nanobiotecnología y la Nanomedicina. De este modo, en la presente tesis se ha estudiado el proceso de biofuncionalización de micropartículas de polisilicio, para actuar como etiquetas celulares, debido al interés que genera la posibilidad de poder etiquetar células vivas y así conocer el comportamiento de las células de manera individual. Sucesivamente, también se ha estudiado la preparación de quimiosensores de dos parámetros intracelulares (pH y calcio) basados en compuestos con capacidad de variar la intensidad de su fluorescencia, según cambios del medio. Concretamente, se ha trabajado en la síntesis e inmovilización de derivados del aminoantraceno en micropartículas de silicio como posibles candidatos para obtener microherramientas capaces de detectar cambios en el pH o en la concentración de calcio intracelulares. Por otro lado, la tesis también describe la preparación de nanosistemas para su aplicación en terapia fotodinámica. La terapia fotodinámica (PDT) se basa en el uso de moléculas específicas (fotosensibilizadores), que en presencia de luz (generalmente un láser), activan el proceso de la muerte celular debido a la formación de radicales libres de oxígeno. La combinación de la utilización de nanopartículas modificadas con un fotosensibilizador resulta un reto interesante que podría mejorar la terapia antitumoral, disminuyendo sus efectos secundarios. Concretamente, en la tesis se describe la preparación de nuevos fotosensibilizadores derivados de porfirinas, con el fin ser incorporados a nanopartículas de óxido de hierro y de oro. También se detalla el estudio de la capacidad de los nuevos nanosistemas obtenidos de producir oxígeno singlete como elemento inductor de la apoptosis celular, y resultados preliminares in vivo indican su potencial aplicación en PDT. Estos estudios, demuestran la posibilidad de dichos nanosistemas para ser usados en terapia fotodinámica. Por último, también se han sintetizado derivados de metalo-porfirinas como componentes de rotores moleculares.[eng] In the present thesis Supramolecular chemistry is exploited to approach applications in the area of Nanomedicine, and it is, focused on the design and preparation of different micro and nanotools for sensing and therapy, in living cells. Initially, the combination of silicon surface chemistry with the incorporation of bioactive molecules has been investigated in order to obtain a potentially microtool suitable for cell tagging. Furthermore, the design and synthesis of organic compounds as intracellular chemosensors was also explored. On the other hand, this report also includes the synthesis and characterization of dissymmetrical porphyrin derivatives and their subsequent incorporation to metal nanoparticles (gold and iron oxide) for their use in photodynamic therapy (PDT), due to their capacity to produce reactive oxygen species after irradiation, inducing the cell death. The preparation of novel metallo-porphyrins as components of molecular machines was also achieved. First, the formation of self-assembled monolayers (SAM) on polysilicon surfaces was investigated, using different silanes to obtain a functionalization protocol which can be easily repetitive and effective. Thus, three silanes with different functional groups, an aldehyde, an epoxide and an activated ester, have been tested to prepare a SAM and subsequently, prompting us to immobilize a bioactive molecule. Different parameters of the functionalization methodology have been examined, such as the silanization time, deposition method, the type of solvent and silane concentration. Once the SAM formation was optimized, the immobilization of the protein what germ agglutinin (WGA) was achieved, because its ability of cell membrane recognition. The WGA used, included a fluorescent dye (Texas red) to be able to characterize the immobilization of the protein on a silicon surface by fluorescence microscopy, and similar successful results were obtained in the three different silanes used. The same methodology (SAM formation and WGA immobilization) was subsequent applied in silicon encoded microparticles designed for tagging cells. Experiments using mouse embryos have been performed to determine the extracellular adhesion level of the encoded microparticles, resulting above 90 % in all cases. Proper immobilization of WGA protein was the key factor in cell labeling, because WGA recognizes specifically certain carbohydrates expressed in the external membrane (zona pellucida) of the embryo. Synthesis and immobilization of an aminoanthracene derivative as pH sensor was carried out, and its subsequent immobilization on silicon microparticles was achieved. Fluorescence spectroscopy measurements demonstrated that the aminoanthracene derivative immobilized on silicon microparticles could be a potential microtool for sense intracellular pH. Fluorescence spectroscopy experiments showed an important increase at acid pH, whereas from pH 7 to pH 12 the fluorescence emission was very low. On the other hand, aminoanthracene incorporating an aza-crown ether was also prepared as a possible candidate for calcium sensing. Preliminary studies using fluorescence spectroscopy, demonstrated a good selectivity for calcium in comparison with other cations such as magnesium, sodium and potassium. Dissymmetrical porphyrin derivatives have been synthesized and then immobilized on gold and iron oxide nanoparticles, obtaining water soluble metallic nanoparticles incorporating the photosensitizer. The capacity to produce singlet oxygen to induce the cell death following irradiation was investigated, resulting porphyrin immobilized gold or iron oxide nanoparticles. Thus, the prepared porphyrin derivatives and their corresponding nanotools exhibited a high formation of singlet oxygen, resulting nanotools potentially suitable for PDT. Otherwise, anti-erbB2 antibody, a specific antibody for a membrane receptor overexpressed in breast cancer cells, was immobilized onto water soluble porphyrin-gold nanoparticles. Preliminary experiments in a breast cancer cell line, demonstrated the capacity of the porphyrin-antibody-gold nanoparticle to produce the cell death following irradiation. Finally a metallo-porphyrins derivative was synthesized and characterize as a promising component for molecular rotors

Supramolecular chemistry for nanomedicine

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/46988Mimicking Nature, supramolecular chemistry represents the chemistry beyond the molecule, in view that intermolecular interactions constitute the driving force for the preparation of molecular and supramolecular assemblies, using the chemical information contained in molecular building blocks. Upon molecular recognition between discrete units, chemical processes such as self-assembly and self-organisation start operating, and are the leading processes to build up supramolecular aggregates and materials. When those materials have dimensions on the nanometric scale, a recently emerging scientific discipline is defined, Nanoscience. Nanomaterials are promising tools for many applications, and their use in biomedical and clinical applications defines the so-called Nanomedicine. In this review we present a few selected examples of nanomaterials designed for therapeutical purposes, emphasizing the importance of the preparation methodology in terms of their therapeutical use

Supramolecular chemistry for nanomedicine

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/46988Mimicking Nature, supramolecular chemistry represents the chemistry beyond the molecule, in view that intermolecular interactions constitute the driving force for the preparation of molecular and supramolecular assemblies, using the chemical information contained in molecular building blocks. Upon molecular recognition between discrete units, chemical processes such as self-assembly and self-organisation start operating, and are the leading processes to build up supramolecular aggregates and materials. When those materials have dimensions on the nanometric scale, a recently emerging scientific discipline is defined, Nanoscience. Nanomaterials are promising tools for many applications, and their use in biomedical and clinical applications defines the so-called Nanomedicine. In this review we present a few selected examples of nanomaterials designed for therapeutical purposes, emphasizing the importance of the preparation methodology in terms of their therapeutical use

Marcatge i identificació directa d'embrions de ratolí mitjançant l'adhesió de codis a la zona pel·lúcida

Due to the increase in the use of assisted reproduction techniques, improvement of sample traceability is essential. As an alternative to current systems of reproductive samples identification, our group work in the development of systems for direct sample codification. In a previous work, we presented a system based on the introduction of polysilicon barcodes into the periviteline space of mouse embryos. Although this system was effective, it showed some limitations. In the present work, we present an alternative approach to overcome these limitations: the attachment of the barcodes to the outer surface of the zona pellucida. This new approach results in an identification rate of 95.6% and this effectiveness is preserved after cryopreservation procedures