Resumen de “multi-functionalization of micro- and nanoparticles for cancer theranostics
This thesis has its main focus on developing multi-functional nanomaterials, that we called nano- and microtools through two different approaches, top-down to provide support and bottom up to give functionality to prepare nanomaterials for diagnosis and therapy in cancer cells (theranostics). It includes functionalization of inorganic and or metallic nano- and microparticles with natural and synthetic receptors capable of acting as sensors to monitor different cellular parameters in living cells and deliverers specifically for diagnosis and therapy in cancer cells (theranostics). For this purpose, we used micro- and nanoparticles as substrates, made up of polysilicon or polysilicon-gold and gold nanoparticles, and functionalized them with (bio)molecules. The first objective was to develop a novel microtool for cell adhesion. For this purpose specially designed polysilicon microparticles of different shapes and sizes were chemically modified, to sense carbohydrates present on tumour cell membranes. An optimized protocol for bio-functionalization of polysilicon microparticles with lectins (WGA and Con A), both on surfaces and in suspension, was developed. Influence of different shapes in bio-functionalization of the microparticles was also observed. The final yield of the number of bio-functionalized microparticles was between 12-21 % with a major loss of approximately 50 % of microparticles during the activation step. These bio-functionalized microparticles in suspension were stable for three consecutive weeks, stored in PBS at room temperature. In vitro experiments were carried out which showed, Con A bio-functionalized Batch 2 microparticles adhered to the membrane of the Dictyostelium discoideum (Dicty) whereas, WGA bio-functionalized microparticles did not adhere to the cell membrane of Dicty or HeLa cells.
Therefore, a synthetic lectin called Boronic Acids (BAs) was used and an optimized protocol for functionalization of polysilicon microparticles with 4-formylphenylboronic acid (PBA), through stable secondary amine bonds was developed. Interaction of BA functionalized surfaces with carbohydrate, N-acetylglucosamine (GlcNAc) was also studied on surfaces using ARS, which indicates stronger interaction between BA and GlcNAc. Polysilicon microparticles of different sizes functionalized using BA showed adhesion to the cell membranes of Dicty and HeLa cells.
In the second objective, the primary goal is to sense intracellular pH in living cells using bi-functional microparticles (polysilicon-gold), in order to differentiate between cancer cells and normal cells. The immobilization of pH dependent fluorophores, Oregon green, pHrodo, SNARF and Alexa fluor on to polysilicon surfaces was achieved successfully. An optimized protocol for the bi-functionalization of two pH dependent fluorophores, Oregon green (on polysilicon) and pHrodo (on gold) on to a hexahedral bi-functional microparticle was achieved for pH sensing.
The third objective was the generation and sensing of Reactive oxygen species (ROS) using a bio-photosensitizer for photodynamic therapy. The selected bio-photosensitizer, Cytochrome c (Cyt c) showed generation of ROS in solution. BODIPY was able to sense the production of ROS from the Cyt c in solution. An optimized protocol for immobilizing Cyt c on to the polysilicon surfaces and BODIPY on gold surfaces and microparticles was achieved. Protocol for bi-functionalization of ROS generator: Cyt c and ROS sensor: BODIPY on bi-functional microparticles was also developed for ROS sensing.
The fourth objective is to deliver anionic drugs using gold nanoparticles synthesized using imidazolium based macrocycles. The ability of these gold nanoparticles to extract and incorporate ibuprofenate from an aqueous phase was calculated to be ca 85 %. The release of ibuprofenate from the gold nanoparticles system follows Fickian diffusion, which could be potentially used for local drug delivery applications.
