Nanopartículas como herramientas en procesos bio-químicos. Síntesis, caracterización, funcionalización y aplicaciones

[EN] `When nature finishes producing its own species, man begins using natural things in harmony with this very nature to create an infinity of species` Jean-Marie Lehn used these words from Leonardo da Vinci to offer his views on the future prospects of supramolecular chemistry . Supramolecular chemistry investigates the principles of nature to produce extraordinary complexes and functional molecular bonds, potentially useful as sensors, catalysts, transportation and other applications in medicine and engineering . Starting with the discovery of the double helix structure of DNA, Biology has grown from being a purely descriptive and phenomenological discipline to a molecular science. All these revolutionary developments have led to merge Biotechnology and Materials Science, giving the undoubted advantage of using biological components to generate new design materials and, conversely, applying these advanced materials and new physicochemical techniques to solve biological problems. In order to exploit and use all concepts involved in natural systems at the nanoscale, development of Nanoscience and Nanochemistry is crucial. Nanoscience is the science that deals with the analysis and manipulation of materials at atomic or molecular level, where features or physical or chemical properties differ significantly from those presented on a larger scale. In this sense, a nanomaterial is defined as a material consisting of a substance or structure having at least one dimension less than 100 nm. These structures have novel properties and a different behavior from the one exhibited by the bulk material of the same composition. Inorganic Nanoparticles are particularly attractive as building blocks for the construction of large superstructures and can be easily prepared from various materials. They have interesting optical, electronic and catalytic properties, which strongly depend on the particle size. During the last twenty years there has been a growing interest in manufacturing inorganic hybrid materials between nanoparticles and biomolecules. As nanoparticles and biomolecules are in a similar scale length, it seems logical that the combination of biomolecules with nanomaterials could lead to some interesting imitation tools of biomolecules, demonstrating the mechanisms of biological processes . At present, it is easy to control and modify the properties of the nanostructures to achieve their integration with biological systems, for example controlling their size or modifying their surface layer to increase their aqueous solubility or biocompatibility. Nanotechnology is defined then as the understanding and control of matter at the nanoscale, at dimensions between approximately 1 and 100 nanometers, where unique phenomena enable novel applications. It is not just a new field of science and engineering, but a new way of looking at and studying. The first practical applications of Nanotechnology were to advances in Communications, Engineering, Physics, Chemistry, Biology, Robotics and Medicine. Nanotechnology in Medicine has been used, and is being used, for the delivery of drugs and developing treatments for a variety of diseases and disorders. The increase in the production of nanomaterials is correlated with subsequent advances in these disciplines. Nanoparticles are used, or being evaluated for use, in many fields, being one of them the field of Proteomics. Nanotechnology in Proteomics has emerged as a promising technological platform for the challenging tasks of studying complex proteomes. Moreover, nanoparticles are a new generation of sensors playing an important role in biomarker discovery. Biomarkers based on interactions with DNA form an important group in the study of, and advances in, the field of cancer and other diseases, and also in the development of treatments with different drugs. Among them, those with fluorescence characteristics are very promising, such that methods aimed at developing them and perfecting them are currently of extraordinary interest. One important field for the use of biomarkers in studies addressing anti-cancer compounds has been opened by the advent of new fluorophores called Quantum dots (QDs). QDs are semiconductor nanocrystals with a size of between 1 and 10 nanometers that are formed by elements of groups II-VI or III-V of the periodic table and that are able to surpass the limits of organic dyes. Their main advantages in this field are as follows: the QD emission spectrum can be modified over a broad range by changing the size and composition of the core, which makes them highly amenable to biological detection. Their broad excitation and narrow emission spectra help to prevent spectral overlapping, thus increasing the possibility of distinguishing multiple fluorophores simultaneously. They also facilitate the use of a single excitation wavelength for QDs of different colours. These properties afford them an advantage over organic dyes, which have narrow excitation and broad emission spectra. The most important aspect to be considered, however, is their capacity for bioconjugation, allowing them to bind to proteins and other biomolecules containing primary amines and carboxylic acid as functional groups, as well as many other substances.There is a broad field of enquiry with many perspectives for the future in the study of QDs for biological applications. Although important advances have been made in certain aspects, there are many weak points as regards the procedures used for the synthesis of these nanoparticles, and their behaviour in different media must also receive more concerted attention. The QDs most widely used as biomarkers, such as CdS, are in most cases synthesized in organic phase, using high-boiling point solvents. The synthesis procedures in aqueous medium are an attractive alternative to the synthesis of QDs in organic medium and are now an active area of interest. Aqueous synthesis shows good reproducibility, low toxicity, low cost and, in particular, the products prepared in aqueous medium have excellent water solubility, stability and biological compatibility. The need to shed more light on the study of their structure and chemical behaviour is crucial if we are to optimize the bioconjugation processes involved, being one of the objectives of this work. Gold nanoparticles (AuNPs) have recently emerged as an attractive candidate for delivery of small drug molecules or large biomolecules (proteins, DNA or RNA) into their targets . Their chemical and physical properties have been exploited for transporting and unloading pharmaceuticals and in photothermal therapeutic contexts. The gold core is inert, non-toxic and biocompatible. They can be easily synthesized in a monodisperse way and core sizes ranging from 1 nm to 150 nm. They have strong and tunable optical absorption. They are easy to modify to endow them with different functionalities, generally through thiol linkages, binding a wide range of organic molecules. Additionally, AuNPs have unique optical properties, such as the characteristic excitation bands in the visible region, owing to the surface plasmon resonance. This property allows the use of AuNPs in many medical, biological and pharmaceutical applications. Currently, AuNPs are attractive candidates for the delivery of a useful load to a particular target site. These useful loads range from being small drug molecules to large biomolecules, such as proteins, DNA and RNA. The efficient release of these therapeutic agents at the appropriate site is a prerequisite for effective therapy and is the basis of directed therapies, mainly in the most advanced fields of medicine. This thesis is divided into three parts. The aim of chapter one is to develop a new process for the preparation of Quantum dots in aqueous medium, in the presence of mercaptoacetic acid (MAA) as a capping reagent, under normal pressure and room temperature. The influence of several experimental variables, including temperature, pH, the Cd/S ratio and the Cd/MAA ratio, on the optical properties of the QDs obtained was studied systematically. The experimental results indicate that these variables play an important role in determining the size and state of the surface of the nanoparticles, and hence their luminescent properties and temporal stability. Aspects like nanocrystal nucleation and growth during the synthesis are important to achieve the best conditions for the synthesis of high quality nanoparticles. A deep study of the physical properties of the nanoparticles and their behavior in solution under different conditions will facilitate their use as biomarkers and in other applications of analytical interest. Chapter two explores the potencial cytotoxic activity of Bile-acid cisplatin derivatives like bisursodeoxycholate(ethylenediamine)platinum(II), PtU2, when conjugated with gold nanoparticles. These derivatives have potential cytotoxic activity and reduced toxicity, owing to their lower lability and their amphipathic character, making them a suitable alternative for CDDP, as anti-tumoral drugs. We analyzed the intracellular delivery ability of these compounds after conjugation with 20-nm gold nanoparticles (PtU2-AuNPs) in the MG63 (osteosarcoma) cell line, a model for one of the most untreatable and painful malignant tumors whose standard treatment requires radical surgery and neoadjuvant therapy. Chapter three is a review about the state of the art of nanoparticles in Proteomics. Protein microarrays are a platform for parallel and simultaneous analysis of protein-protein interactions and protein profiling. Together with the advances in microarray technologies, increasingly sensitive and reliable detection methodologies are being currently developed. Such protein detection systems have progressively undergone a relevant transition from label-based to more sensitive label-free technologies, which are extremely useful to study the interactome and functions of large amounts of proteins on demand. Overall, label-based systems are mainly focused on the use of specific tags for target molecules as conventional fluorescent dyes and radioisotopes, among others. More recently, other substances are being proposed, including inorganic quantum dots (QDs), gold nanoparticles (NPs), Raman dye-labeled carbon nanotubes or silica NPs. On the other hand, label-free detection techniques include surface plasmon resonance (SPR), carbon nanotubes, cantilever, etc. The successful coupling of nanotechnology with proteomics over the last few years has led to the development of nanoproteomics, which provide a robust analytical platform for real-time and sensitive detection of low-abundande proteins. A number of nanotechniques have been lately used for diverse applications such as biomarker discovery, label-free protein detection, study of protein interactions and printing of protein microarrays. Among all nanomaterials, Quantum dots, gold nanoparticles and carbon nanotubes have demonstrated potential to overcome the challenges of sensitivity faced by conventional proteomics for biomarker detection. However, many efforts are still required to explore the toxicity and biocompatibility of nanotechniques to ensure their safety for biological applications.

Author

• Sánchez Paradinas, Sara

• Tesis y disertaciones académicas
• Universidad de Salamanca (España)
• Tesis Doctoral
• Academic dissertations
• Bioquímica
• Química analítica
• Biochemistry
• Analytical Chemistry

Identifier

hdl:10366/123068

OAI identifier oai:identifier

oai:gredos.usal.es:10366/123068