Resumen de Synthesis and functionalization of nano- and micro-particles for sensing and therapy in living cells
Oriol Penon Esteva
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.
