Design and development of specific nanostructured systems based on biocompatible materials

  1. Hassan López, Natalia
Dirixida por:
  1. Juan Manuel Ruso Beiras Director

Universidade de defensa: Universidade de Santiago de Compostela

Fecha de defensa: 07 de decembro de 2011

  1. Víctor Mosquera Tallón Presidente
  2. Angel Piñeiro Guillén Secretario
  3. Julia Maldonado Valderrama Vogal
  4. Sergio Moya Vogal
  5. Laurent Bouteiller Vogal

Tipo: Tese


The different applications and uses of proteins have grown over the past 50 years continuously. However, in recent years we have been witnessed an overwhelming appearance of cutting-edge applications, biommimetic materials, tissue engineering, drug delivery, bioelectronics and nanomodels. In this sense, the interactions between small molecules in solution of proteins affect their respective function and determine the stability. Biological relevance of the solution with respect to aggregation, liquefaction and other phase transformations. In addition, protein aggregation, crystal formation, folding ot denaturation are defined largely by the forces acting on the molecules. Proteins play a fundamental role in the structure and function of biological cells and organism. They are structurally organized by the binding of different amino acids that have hydrophilic or hydrophobic groups, for this reason proteins are considered as macromolecular surfactants due to amphoteric property. Protein structures contain four levels of complexity: primary, secondary, tertiary and, sometimes but not always, quaternary. Each level has its own characteristics, and all levels are related to each other and depend on each other, together creating an extremely complex network. The primary structure is built up by the linear sequence of amino acids as joined together by peptide bonds (and includes any disulfide bond). The peptide bond is a chemical, covalent bond formed between the ¿-amino group of one amino acid and the ¿-carboxyl group of another. Disulfide bonds are often present in extracellular proteins, but are rarely found in intracellular proteins. These are primarily disulfide bonds between cysteine residues that are adjacent in space but not in the linear amino acid sequence. Amino acid compositions vary enormously from protein to protein. Except in certain special cases, the various amino acids are distributed apparently randomly along the polypeptide chain. All properties of a protein are derived from the primary structure, the linear sequence. They may be classified into three main types: fibrous, globular and disorderer. The secondary structure in a protein is the regular folding of regions of the polypeptide chain. The two most common types of protein fold are the alpha-helix and the alpha-sheet. In the rod-like alpha-helix, the amino acids arrange themselves in a regular helical conformation. The alpha-sheet structure is a pleated sheet composed of beta-strands in parallel or antiparallel arrangement depending on whether they run in the same direction or in opposite directions, respectively. The secondary structure elements fold into structural units, called domains, which comprise the tertiary structure. Tertiary structure refers to the spatial arrangement of amino acids that are far apart in the linear sequence as well as those residues that are adjacent. This structure is maintained by four types of interaction between side chain groups of amino acids residues: hydrogen bonding, ionic interaction between oppositely charged group, hydrophobic interactions and disulfide cross-linkages. Proteins containing more than one polypeptide subunits exhibit quaternary structure, referring their arrangement in space. The interaction of small molecules (surfactants, drug molecules and denaturants) with macromolecules with specific receptors sites on surfaces of supramolecular organizations of biological systems in one of the extensively studied phenomena in recent biochemical research as it plays a role in a vast range of vital biochemical phenomena. Proteins interact strongly with oppositely charged surfactants in aqueous solution due to a hydrophobic attraction between the surfactant tail and hydrophobic region on the surface and in the interior of the protein, as well as an electrostatic attraction between the headgroup of the surfactant and the protein (amphiphilic molecules). Many pharmacologically active compounds are amphiphilic molecules, and exhibit the same behavior as traditional surfactants, i.e., they tend to self-associate to form micelles when dispersed in aqueous solutions. Amphipatic molecules such as surfactants interact with proteins and to alter their structure, physico-chemical and rheological properties. Therefore, understanding of interaction between the surfactants and proteins in the bulk and the interface, formation of protein-surfactant complexes and displacement of protein molecules from the interface by surfactant molecules is important from scientific as well as practical viewpoints. With anionic surfactants, the interaction is predominantly electrostatic, and exist different models that explain how anionic surfactant interact with proteins. Compared to the anionics, cationic surfactants weakly interact with the proteins as a consequence of smaller relevance of electrostatic interactions at the pH of interest. However, the binding isotherms of both types of surfactants have been found to be similar. The surfactant ions bind to groups of opposite charge on the protein and hydrophobic interactions between the aliphatic chains of the surfactants and the non polar protein surface regions that are adjacent to charged sites occurs. These initial interactions cause the protein unfold, which result in the exposure of more binding sites, and, as the surfactant concentration is increased, binding becomes cooperative, and ultimately saturation occurs. Compared ionics, nonionic surfactants bind weakly to the proteins due to the absence of electrostatic interactions, thus making micelle formation in bulk more favourable. Both, proteins and surfactants have the quality to adsorb on an extensive range of surfaces, hydrophilics or hydrophobics Proteins and low molecular weight surfactants can interact in the bulk and at the surface in different ways, resulting in complexes with different surface activity. Proteins and surfactants stabilize interfaces through different mechanisms. Surfactants form mobile surface layers which are stabilized by the Gibbs-Marangoni mechanism. In contrast, proteins stabilize interfaces by forming a strong viscoelastic network through electrostatic, hydrophobic, and covalent interactions with neighboring proteins. These two mechanisms can be incompatible, and thus, the study of the specific interactions occurring between proteins and surfactants and the characterization of the interfacial structures is essential for rational manipulation of interfacial structures for future technological applications A general model explaining how anionic surfactants interact with globular proteins has been suggested by several authors. The model comprises three stages: specific binding, non cooperative binding and cooperative binding. In the first stage, the surfactant binds with specific sites on the surface of the protein. Ionics bonds are formed between the charged groups of the ionic surfactants and the opposite charged amino acid residues of the protein. Hydrophobic interactions between the aliphatic chains of the surfactants and the nonpolar protein surface regions that are adjacent to charged sites are also observed. On the other hand, exist models that also take into account not only ionic and hydrophobic interactions, but also hydrogen bonds between the oxygen groups of the surfactants and the nitrogen groups of the peptide linkages. In the second stage, the tertiary structure of the protein unfolds, either as a result of electrostatic repulsion between the charges of the surfactant bound to the protein, or because the hydrophobic chains of the surfactants penetrate the apolar regions of the protein. Hydrophobic interactions then take place between the chains of the surfactant molecules and the new nonpolar residues of the protein that are exposed denaturation proceeds. This occurs when the concentration of the surfactants approaches the cmc. The pH has also an effect on the net charge of the protein, which depends on its isoelectric point (pI). Below pI (when the net charge in aqueous solution is zero), the protein has a positive net charge, and interactions with anionic surfactants generally results in precipitation of the complexes, owing to neutralization of the charges. Above pI, the negatively charged protein forms stable soluble complexes. To a first approximation, protein solubility in water increases with the proportion of polar and charged groups, and decreases with increasing molecular weight. Most protein show a minimum in solubility at the isoelectric point where electrostatic interactions are minimal. Amphiphilic molecules are used widely in both consumer and industrial applications such as food processing, medicines and pharmaceuticals. Because of their peculiar self-assembly behavior, their properties have evolved from being of a purely scientific interest to become a key concept in nano and biotechnology applications, such as drug delivery, sensors, and catalysts. The design of drugs must take into account several attributes, such as the size of the encapsulation system and stability. For applications in drug delivery, previous reports emphasized that the distribution of nanoparticles in tissues and organs is a function of size. In addition, nanoparticles must be sufficiently stable (able to with stand mechanical stress) to increase circulation time in blood and with adequate thermal stability to reduce side effects. Thus, molecular architecture should be involved in the design of self-assembling systems for preparation of nanoparticles. Moreover, the adsorption of proteins onto a biomaterial surface, and consequently, the nature of the adsorbed protein layer is crucial element of biological response. The study of the interactions of blood proteins with implanted biomaterials has recently been recognized as one of the most challenging for the new generation of biocompatible materials. Recent advances in nanotechnology or hybrid nanomaterial have triggered the enthusiasm for these complexes and systems. For this purpose the main aim of this thesis is to design biocompatible materials for different applications, specifically drug delivery. Firstly, we have characterized the self-assembly of surfactant and drugs, and its interaction with proteins such as mioglobin, lysozyme, ovalbumin and fibrinogen. Finally, we have prepared nanomaterials to adsorb drugs and we studied its interaction with proteins. In the compilation of this thesis an attempt has been made to give appropriate recognition to the current interest in normal and emergency applications of these systems, by discussing such aspects as newer systems, unusual approaches and highly used techniques including information about the physical principles and effectiveness of selected techniques. For this reason it has been focused on the experimental point of view. 1. Study and characterization of different amphiphilic molecules Firstly, it has been studied and characterized several amphiphilic molecules, as well as the characterization of mixtures of heterogeneous molecules. Different experimental techniques have been used, such as electrical conductivity, density, ultrasound, dynamic light scattering, nuclear magnetic resonance spectroscopy, molecular dynamic simulations, transmission and cryo-transmission electronic microscopy, confocal microscopy and UV-Vis spectroscopy. From these techniques and theoretical results we have studied the BTS self-aggregation process. Such association seems to be entropically driven, as indicated the thermodynamic analysis. The logarithm of the CAC as a function of temperature is U-shaped, in agreement with most amphiphilic drugs. The aggregate formation in absence of salt is related to a likely larger ionization degree and a better ability to form hydrogen bonds. Nevertheless in the presence of salt the higher degree of salt ions binding on the aggregate surface reduces the repulsive interaction between head groups, thus promoting the aggregates growth. In addition, apparent coefficients of expansibility as well as compressibilities indicate that the aggregation process tends to decrease the intramicellar repulsion, in the presence of salt, due to the shield of the ionic interaction. This process could be explained by the presence of steric interactions producing negatives apparent molar adiabatic compressibility values. Based on the similarities of our volumetric results with other drugs molecules, a BTS arrangement with the aromatic rings toward the core of the structure upon aggregation has been proposed. This fact is supported by the analysis of NMR spectra and molecular dynamics simulations. The size of the self-associated nanoparticles was estimated to be about 2.5-3.0 nm with an aggregation number of ~30 BTS molecules. Is very important to note that from experimental techniques and dynamic molecular simulations we have studied mixtures of heterogeneous molecules, as well as the synergism established between these molecules. Our results show that equimolar mixtures of the short anionic sodium perfluorooctanoate (PFO) molecule with the twice longer canionic cetyltrimethylammonium bromide (CTA) in the absence of counterions may form different structures at different concentrations in the bulk aqueous solution. The higher the catanionic surfactant concentration, the higher the density of the solution and the lower the compressibility, the available volume per solute molecule, the area exposed to the solvent, and the diffusion coefficient of both PFO and CTA molecules. Specifically, DLS experiments indicate that at least two populations of structures exist in the studied concentration range. While the structures present in the first population grow up with the surfactant concentration, the size of the structures in the second population, which were identified as vesicles, is practically constant with a radius of ~80 nm. At high surfactant concentration both populations tend to converge to these vesicles which were observed by TEM, cryo-SEM, and confocalmicroscopy in the same concentration range. The spontaneous formation of unilamellar homodispersed vesicles has already been observed in the literature for similar systems. Lamellar phases, also common in hydrogenated catanionic systems, were not found under the studied conditions. Molecular dynamics simulations allowed to observe how equimolar mixtures of PFO and CTA molecules initially located at random positions in water also form different structures, including vesicle patches, within time scales shorter than 20 ns. The variety of structures observed is ascribed to the balance between the different interactions occurring between PFO and CTA as well as with the solvent molecules. The well known dual lipophobic/hydrophobic character of fluorocarbon molecules is expected to be an important contribution for the aggregation of this catanionic surfactant. Using a similar force field parametrization in nonionic fluorocarbon-hydrocarbon diblocks we recently observed the spontaneous formation of fluorine-rich and hydrogen-rich domains at the solution/air interface by MD simulations. The lack of fluorinated/hydrogenated domains in the nanostructures obtained in the present work indicates that the electrostatic interactions are dominant in these systems. The increase in the PFO/CTA contact area when the surfactant concentration rises, supports this conclusion. Interestingly, since the length of the CTA molecule is twice that of the PFO molecule, the former does not fit well in the structures and the end of its chain forms a hydrocarbonated core in the aggregates. Unfortunately, our atomistic MD simulations did not allow observing the formation of entire vesicles. Overall, this work shows the versatility of these molecules to form a variety of structures due to the balance between their different lengths, the electrostatic interactions between their heads with charge of different sign, and the particular interactions occurring between fluorinated and hydrogenated carbon chains. In addition in this thesis we proposed the new idea of ``self-assembled drug¿¿ based on the combination of two or more drugs. As model system we used hexadecyldimethylammonium bromide: dicloxacilin at room temperature. The two drugs are well known for their therapeutic effect and together form wormlike micelles with the combined therapeutic effect of both individual compounds. The system was investigated using, density and sound velocity, dynamic light scattering and cryo- electron microscopy (cryo-TEM) and UV for a complete physicochemical and stability characterization. This is a first step for further research about their therapeutic activity and the efficiency compared with other classical delivery methods using drugs embedded in vectors. Many other self-assembled drugs like vesicles and liquid crystals can be obtained by the appropriate combination of single therapeutic compounds. We expect our work to encourage the scientific community to investigate new self-assembled drugs and their advantages over more conventional strategies in drug delivery. 2. Energetic study of interactions between protein-drugs and structural analysis of complex formed. We have analyzed complex formed between proteins and amphiphilic molecules. For this type of study we used, in addition to already mentioned, some techniques, such as circular dichroism (CD), differential scanning calorimetry (DSC), surface tension, atomic force microscopy (AFM), rheology. We have obtained relevant results. As previously we studied the self-aggregation of BTS, we have studied the effect of this molecule in thermal stability of lisozyme, mioglobina, ovalbumina, and fibrinogen. All the proteins had different denaturation temperatures, indicating differences in thermal stability. The BTS binds though electrostatic interactions to myoglobin and lysozyme, and through hydrophobic interactions to ovalbumin and fibrinogen. We have observed that higher concentrations of BTS decrease Tm and favour protein unfolding. A major effect is observed in the case of myoglobin, the protein with the highest ahelical secondary structure (75%). This could be related with the fact that backbone hydrogen bonds of a-helix are generally slightly weaker than those found in b-sheets. Thus, they are readily attacked by the surrounding BTS molecules. Relevant results also were obtained with fibrinogen and acebutolol or propranolol. It was found that these molecules plays two opposites roles in the folding and stabilizing the end D fragments of fibrinogen: promotes stability at low concentrations and unfolding at the higher ones. With the other domains along the protein structure, acebutolol and propranolol act as potent denaturant for C-terminal of the A¿-chains and denaturant at higher concentrations for the central E domain. Transitions from the native to the unfolded state of fibrinogen induced by these molecules have been found to be a multiple step process with intermediate molten globule states. It was also observed that the presence of acebutolol and propranolol causes an increase in the content of b-sheets at the expense of a-helix structures at lower temperatures. It is postulated that fibrinogen form dimmers in the absence of additives due to electrostatic interactions between the negative domains (D and E) of one molecule and the positive domains (C) of the another. The addition of small amounts of acebutolol results in the aggregation of the dimmers to tetramers dissociate to dimmers. These findings could be useful for applications in biomaterial science where devices should be created with improved hemmocompatibility. Also, we studied the supramolecular aggregation between fibrinogen and fluorinated and hydrogenated surfactants. As a first step, we have studied the thermal stability of fibrinogen in the presence of the surfactants. Addition of hydrogenated surfactants results in decrease in melting temperatures. However, the fluorinated plays two opposite roles in the folding and stability of fibrinogen: acting as a structure stabilizer at low molar concentrations (enhancing Tm) and as a destabilizer at higher concentrations (diminishing Tm). Unfolding process of fibrinogen does not follow a two-state process but involves intermediate states for all studied systems. Increasing temperature and/or surfactant concentration results in a decrease in ¿-helix (and ¿-sheet increase) content. However, both the quaternary and tertiary structure does not undergo large variations as can be inferred from UV¿vis and Raman spectra. SAXS measurements have shown that pure fibrinogen exists as a paired-dimer in this medium. In the presence of surfactant, the map of the configurations of the protein changes, depending on the hydrophobicity of surfactant. The presence of C8HONa (lowest hydrophobicity) did not promote any significant change in fibrinogen. C8FONa monomers interact with fibrinogen paired-dimer without promoting dissociation or significant conformational changes. On the other hand, the interaction between C12HONa (highest hydrophobicity) and the protein promotes the surfactant self-assembling at hydrophobic moieties. Finally, we would like to point out that the picture of the fluorocarbon/hydrocarbon plus fibrinogen system reported here could provide a key that paves the way for future biochemical and biomedical applications, for example, in the recovery of proteins and protein conformation on support materials for regenerative therapies. For this reason we have characterized the surface of these complexes. The present surface tension measurements suggest complex formation of fibrinogen with the fluorinated surfactant, as indicated by the slight displacement to lower concentration of the cac with respect to the value for the pure surfactant. For fibrinogen/hydrogenated surfactant mixtures, the cac coincides with the cmc of the pure surfactant. The interaction between fibrinogen and fluorinated surfactant causes a strong cooperative or synergistic effect resulting in an increased surface activity of the mixed system relative to fibrinogen. Fibrinogen seems to prevent the formation of a cohesive structure upon adsorption with a low surface dilatational modulus, and in all cases, addition of surfactant results in a reduction of the elasticity of the surface layer accompanied by an increase of the surface pressure. However, in the case of fibrinogen/hydrogenated surfactant mixtures, the elasticity of the surface layers only diminishes over a certain concentration of surfactant. Thus, based on this fact, and on the absence of interaction of fibrinogen/hydrogenated mixtures, compaction on the surface layer could be expected. AFM images are consistent with the surface characterization studies. Formation of clusters in the case of fibrinogen/fluorinated mixtures is consistent with results inferred from surface characterization. In the case of fibrinogen/hydrogenated surfactant mixtures, no cluster formation is seen; indeed, the size of the individual molecules is similar to that of the pure FB system. These observations are consistent with the results reported from the dilatational moduli data. We have studied the hydrogel formation with the aim to synthesized mesoporous materials. In this work, we have provided some novel insight about the gelation of ovalbumin¿surfactant mixtures. The main features from this study can be summarized as follows. Firstly, gelation of ovalbumin¿surfactant systems for three different surfactants (C8HONa, C8FONa and C12HONa) at different surfactant concentration mixture ratios in the range 0¿10mM was studied, and the strength of the gel was observed to increase with increasing surfactant concentration. For the three surfactant under study gel strength follows the order C8FONa > C12HONa > C8HONa. Secondly, the surfactant concentration dependence of the storage modulus can be described by power laws. The results suggest a fibrillar structure of the gels and a fractal dimension dependent of the surfactant nature. Finally, based on viscosity measurements, the different nature of the fluorinated surfactant provokes a disruption of the gel network at high concentrations. 3. Structural and energetic characterization of the templates obtained by different strategies: In this study, we have designed a simple and controllable route for the synthesis of opals-CT materials with unusual fibrous microstructure similar to those existed in the nature using a bottom-up microemulsion droplet system as chemical microreactor. Themicrocrystalline structure of opals and consequently their optoelectronic properties are a result of a particular combination of all the relevant microemulsion parameter, hydrothermal treatment time, and calcination temperature. The obtained fibrils are longer than 20 ¿m with a diameter of 30-50 nm and are clustered forming bundles of 100-200 nm distributed in different orientations. Fibrils are composed by RSiO2 polyhedral spheres that are monodisperse in size (d ¿ 2 Å) packed, as usual in photonic crystals, in (111) hexagonal layers superimposed on one another along the [111] axis to form a 3D fcc lattice. The lattice constant (a = 2.83 Å) is on the order of angstroms as for ordinary crystals and very inferior to those obtained until now for synthetic opals. Because of their unusual microstructure, they exhibited short and long wavelength photolumiscence emissions which differ according to the crystobalite-trydimite stacking. The material with high content of trydimite-type stacking faults shows high intensity ultraviolet A and B just with minor intensity bright-red and yellow emission, while the material that presents a high ordered script of cristobalite emits ultraviolet A, orange, and green light radiations. It is believed that a combination of a quantum mechanism and the presence of defective Si oxide layers are the responsible for the simultaneous emissions of S- and F-bands. Additionally, the computed band gap values (5.50 and 4.41 eV) for both synthesized materials are similar to those obtained for silicon-based metal oxide semiconductors and highly inferior to the experimental band gap values obtained for pure crystalline SiO2 polymorphs (8.9 eV).