Design and development of specific nanostructured systems based on biocompatible materials

Soft Matter and Molecular Biophysics Group Department of Applied Physics Faculty of Physics Doctoral Thesis, 2011 DESIGN AND DEVELOPMENT OF SPECIFIC NANOSTRUCTURED SYSTEMS BASED ON BIOCOMPATIBLE MATERIALS Natalia Hassan López Facultad de Física-Campus Vida 15782 Santiago de Compostela D. Juan M. Ruso Beiras, Profesor Titular del Departamento de Física Aplicada de la Universidad de Santiago de Compostela, INFORMA: Que el trabajo de investigación titulado: “Design and Development of Specific Nanostructured System Based on Biocompatibles Materials” que presenta la alumna Natalia Andrea Hassan López ha sido tutorado bajo mi dirección dentro del grupo “Materia Blanda y Biofísica Molecular” de la Universidad de Santiago de Compostela y reúne los requisitos de calidad y rigor científicos necesarios para optar al Grado de Doctor en Ciencias Físicas. Para que así conste a los efectos oportunos. Santiago de Compostela, septiembre de 2011 Juan Manuel Ruso Beiras To my parents Acknowledgments It not easy to leave home and leave family, but I am very sure that It was the most comfortable and rewarding experience in my life. I am very grateful to have met very special people in this magic city, Santiago de Compostela. Firstly I would like to thank to my supervisor Juan Ruso who was the most important person in this thesis. He gave me the scientific support and I appreciate all his contributions of time, ideas, and funding my PhD experience productive and comfortable. The joy and enthusiasm he has for his research was contagious and motivational for me. Thank you Juan for trust me and give me this opportunity to live the most important experience in my life. I also want to thank all the group members of Soft Matter and Molecular Biophysic group that I have worked, Ángel Piñeiro, Pedro Vazquez, Paula Messina and Valeria Verdinelli. During this work I have had the pleasure to work with Victor Morris, Patrick Gunning and Julia Maldonado-Valderrama in the Institute of Food Research (Norwich-UK). For this reason I would like to thanks these persons for this incredible opportunity to learn, deepen my knowledge and for everything you contributed to my stay. I wish to express my sincere thanks to Josué Juarez, one of the most important friendlabmate during these years, for his patient, enthusiasm and immense knowledge: Thank you for all “Wua yu sei”!. Also, I wish to thanks to all wonderful people, my special friends of the faculty: Irais Bautista, Elena Blanco, Paula Toimil, Miguel Morales, Antonio Topete, Alejandra Flores, Ceila Fong. All of you were a support and were very important part of my life these years. Last but not the least, I would like to thank my family: My father Hector Hassan, my mother Viviana López, my middle sister Marjorie Hassan and my youngest sister Belén Hassan for their unconditional love and their immense support in all my decisions. These years have not been easy for us far from home, but I am very grateful for their patient, I love you so much. Abstract The challenges and applications in the nanomaterials research domain are numerous: medicine, energy, biosensing devices, technology. This motivated us to address in this study new design strategies and concepts to develop new biofunctional materials to achieve bioand hemocompatibility. This thesis is divided in three main parts. The first part has been focused on the self-assembled of surfactants and drugs where has been found to depend on several factors, such as hydrophobic chain length and type, temperature, the nature of contraion, etc. As well as we have studied the effect of mixing alkanes and perfluoroalkanes and a new idea of “selfassembled” drugs based in the combination of one or more drugs. We have focused on how the principal experimental parameters are determined and how these data can be evaluated to obtain relevant information of the interaction. Different techniques have contributed to a deeper understanding of interactions between proteins and surfactants/drugs. The complexation between serum proteins and these different surfactants and drugs has been analyzed at different conditions. We have analyzed conformational changes as a function of different external agents and as well as the stability of the complexes and the stability of the complexes besides. Then, we designed optimal templates that manage the formation of biocompatible materials. For this purpose we used reverse microemulsions and proteins. We have determined structural properties (shape, size, stability) as a function of different external parameters. In addition, this characterization has been realized with experimental and dynamic molecular simulations. Both of them allowed us to optimize the design process of templates with specific purposes. The final part of this thesis is devoted to prepare the materials with structures not studied in details currently; with the purpose to obtain stable materials and with biocompatibility properties. The last step was to evaluate the adsorption of proteins and drugs, characterized in the first part, in specific conditions. The structure of this thesis has been presented distributing the work in four different chapters based on homogeneity and steps to reach the aim. Each chapter has been structured by a brief introduction where the subject is exposed as well as some important facts or descriptions of concepts or techniques. The relevant aspects of the research are exposed and finally the publications that have been obtained are appended. Chapter 1: INTRODUCTION 1. Introduction Amphihilic compounds, that is, those that have distinct hydrophilic and lipophilic parts, are used in most branches of industry and are ubiquitous in biological systems. They range from low molecules weight substances (like surfactants and lipids) to macromolecules (comprising synthetic graft and block polymers) and biomacromolecules (like proteins, lipopolysaccharides, and nuclei acids)[1-3]. Amphiphilic molecules self-organize both in bulk solution and at interfaces. Low-molecular-weight amphiphilic compounds, mainly constituted of surfactants and polar lipids, have been thoroughly investigated extensively and are well understood both with respect to their bulk self-assembly and surface modifying ability. The study of high molecular weight amphiphilic molecules is much more recent. Regarding the effect that these compounds have on their interactions with biomacromolecules it has been proposed that the amphiphilic behavior of these molecules play an essential role for attachment at the polipepetide chain, leading to their high efficiency[4]. Proteins are not only the building devices of life, but also rule life. They play a variety of roles in life processes: they are structural proteins (viral coat proteins, epidermal keratin); catalytic proteins (enzymes); transport and storage proteins (haemoglobin, myoglobin); regulatory proteins (hormones); and proteins of the immune system and the immunoglobulin superfamily. Much biotechnological, medical and drug delivery system aims to predict their behavior on the basis of the set of molecules involved, mainly proteins and amphiphilic molecules, specifically drugs. Understanding the interactions between these molecules is therefore crucial to such efforts. Although many thousands of interactions are known, precise molecular details are available for only tiny fraction of them. The difficulties that are involved in experimentally determining atomic structure for interacting proteins make predictive methods essential for progress. Structural details can ultimately turn abstract system representations into models that more accurately reflect biological reality[5]. Proteins undergo changes in their natural state by the action of ligands, which after their adsorption onto the protein can control the hydrophobic-hydrophilic character of protein surface. Understanding protein-ligand interactions is central to discovery new medicines, and, when the ligand is a drug, these interactions are central to drug delivery to benefit human health. Essential for high biological activity are a good geometric fit and a high degree of complementary of hydrophobic and polar parts of both entities, the binding site of the protein and the ligand. 19 On the other hand, the synthesis of nanoparticles and nanoporous materials is a recent branch of the nanotechnology. Increasingly, colloidal chemists are contributing to the biomimetic synthesis of inorganic nanostructures with dimensional, morphological and architectural specificity by using organized self-assemblies of surfactants as nanostructured reaction media or templates. A large variety of methods have been proposed to synthesize nanoparticles, among them the use of microemulsions enables the control of particles size, as well as protein hydrogels as a templates. Also these bio-inspired templates can offer a fast, general, inexpensive and environmental friendly procedure for the synthesis of nanostructured materials[6-8]. The reduction of such materials at the nanometer scale offers a number of outstanding optical and mechanical properties, including electrical, magnetic, thermal, chemical and biomedical properties. 1.1 Aim 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. 20 Chapter 1: Introduction The specific aims are: 1. First Aim: Study and characterization of different amphiphilic molecules (BTS, acebutolol, propranolol, C8HONa, C8FONa, C12HONa, dicloxacillin, CTAB) estimating the critical micellar concentration including the thermodynamics properties of micellization, as a function of temperature, concentration, and also salt concentration. As well as the characterization of mixtures of heterogeneous molecules (dicloxacillinCTAB, C8F--CTA+) which self-assemble in aqueous solutions and to propose a new idea of self-assembled drug based on the combination of two or more drugs. Study of protein-drug interaction, structural analysis of complex formed. Influence of concentration, temperature and environment. Interaction protein-drug by fluorescence, RAMAN spectroscopy, UV-Vis, differential scanning calorimeter (DSC). Characterization of complex protein-drug by dynamic light scattering (DLS), circular dichroism (CD), RMN, Transmission Electron Microscopy (TEM), SAXS. Achieving this objective will know the interactions between the most important proteins and drugs selected. Also will be characterized the structures of the complex formed as well as conformational changes in the protein induced by drugs. This knowledge will design the subsequent study: 2. Second aim: Structural and energetic characterization of the templates obtained by different strategies: A) Fibrinogen hydrogels B) reverse microemulsions of aerosol OT (dioctyl sodium sulfosuccinates), phase diagrams of template (cubic, lamellar, hexagonal) depending on the length of the alkyl chain (4-16), electric charge (pH 3,2) and temperature range (15-45°C) with measures of electrical conductivity, Differential Scanning Calorimeter (DSC), Transmission Electron Microscopy (TEM). Study of colloidal stability of the systems by processing data from dynamic light scattering (hydrodynamic radius). Study dynamic surface tension in different media and temperatures. Structural analysis of the systems by TEM and rheology. Study of the AOT microemulsion / oil phase / water as a function of temperature, different oil phases (hexane, n-heptane, cyclohexane), pH, ionic strength. Characterize by dynamic molecular simulations the behavior of certain samples of elements of the templates at diverse concentrations and temperatures. Achieving this goal will be able to determine the structure, chemical stability and functionality of the templates based on specific use. Chapter 1 Introduction 21 3. Third aim: Preparation of mesoporous materials, adsorption of active principles and study of the delivery from the material. Design and characterization of the pores of the ceramic material (TEOS, SiO2). Determine the number, size, shape, distribution, connectivity and possible functionalization of their walls, depending upon the drug and the ceramic material is to be used (both pure and mixtures in various proportions) based on nitrogen adsorption measurements and SAXS, SEM, TEM, IR. 22 Chapter 1: Introduction Chapter 2: METHODS 2. Methods 2.1 Introduction There is a wide range of methods available to characterization of different colloidal particles. To establish these methods it is necessary to take into account some considerations, that is, it is possible to define the following groups: • Physical Chemistry Characterization • Electrical and spectroscopic properties • Size and shape distributions • Concentrated solutions: Ordering and structural organization To study the physicochemical properties of soft materials it is possible to use several techniques, which in the case of surfactant molecules, can provide us information related with self-aggregation partnerships, energy, interactions between aggregates, interactions with surrounding media or stability. 2.2 Conductivity Conductivity is a physical property using for studying ionic solutions. The conductivities have been extensively used mainly for determining the critical micelle concentration (cmc) of ionic surfactants, at which the concentration dependence of each conductivity breaks. Along the definition of cmc by Philips in which the cmc is considerate as the concentration at which the slope of the plot of a physical property of the solution against concentration is changing most rapidly. The conductivity allows us to determining ionic constants of aggregates such as equilibrium constant, degree of counterion dissociation of micelles and aggregation number. The conductivity of a solution depends on the transport of charges. Species with a small hydrodynamic radius conduct better than larger ones since they can move fast. Ions of higher charge usually attract more water, resulting in a larger hydrodynamic radius. For ionic species 25 The conventional or single-beam spectrophotometer contains two source lamps, a deuterium lamp and tungsten lamp. Polychromatic light from the source is focused on the entrance slit of a monochromator, which selectively transmits a narrow band of spectrum. This light then passes through the sample area to the detector. The absorbance of a sample is determined by measuring the intensity of light reaching the detector without the sample (the blank) and comparing it with the intensity of light reaching the detector after passing through the sample[11]. When the light passes through or is reflected from a sample, the amount of light absorbed is the difference between the incident radiation (I0) and the transmitted radiation (I). The amount of light absorbed is expressed as either transmittance or absorbance. Transmittance usually is given in terms of a fraction of 1 or as a percentage and is defined as follows: 𝑇=𝐼 𝐼0 𝑜𝑟 %𝑇= (𝐼 𝐼0 ⁄) × 100 (2.9) Absorbance is defined as follows: 𝐴=−𝑙𝑜𝑔𝑇 (2.10) For most applications, absorbance values are used to determine many physicochemical characteristics of compounds and thus can provide information as to identify of a particular compound. For example the absorbance spectra of proteins result largely from the presence of the aromatic amino acids tryptophan, tyrosine and phenylalanine. A protein at room temperature has a specific tertiary structure or conformation that in turn creates a specific electronic environment for the aromatic amino acids. If the protein is heated it will, at certain temperature, unfold or melt and lose the structure. In this process, the electronic environment of the aromatic amino acids changes, which in turn results in spectral changes or shifts. However, the observed absorbance is always significantly less than expected because their electronic environment. When a molecule is heated, the hydrogen bonds break, the double helix unwinds, and the absorbance increases. 32 Chapter 2: Methods 2.8 Fluorescence Fluorescence is the process by which electronically excited molecules decay to the ground state via the emission of a photon without any change in spin multiplicity. Emission is detected by spectrofluorometers and occurs at longer wavelengths than the corresponding absorbance band. The quantum yield of fluorescence emission is defined as the ratio of photons emitted through the fluorescence to the number of photons absorbed. Changes in intensity and wavelength reflect the influence of local environment on emission. The quenching of fluorescence is known to occur by two process, namely collisional (dynamic) quenching and/or formation of a complex between the quencher and fluorophore (static quenching)[12]. The fluorescence quenching data were analyzed according to the SternVolmer equation: 𝐹0𝐹 ⁄= 1 + 𝐾𝑠𝑣[𝑄] (2.11) where 𝐹0 and 𝐹 are the fluorescence peak intensities of the fluorophore in the absence and presence of quencher. Collisional quenching can determine the accessibility of fluorophores in proteins via the Stern-Volmer analysis. When the drug molecules bind independently to a set of equivalent sites on a protein, the equilibrium between free and bound molecules is given by the equation: log �𝐹0−𝐹 𝐹�=log 𝐾+𝑛log[𝑄] (2.12) where 𝐹0, 𝐹, are the stady-state fluorescence intensities in the absence and presence of quencher; 𝐾 and 𝑛 are the binding constant and the number of binding sites, respectively. Chapter 2: Methods 33 2.9 Light Scattering Dynamic Light Scattering (DLS) is also known as Photon Correlation Spectroscopy. This technique is one of the most important methods used to determine the size of particles. When a beam of light passes through a colloidal dispersion, the particles or droplets scatter some of the light in all directions. When the particles are very small compared with the wavelength of the light, the intensity of the scattered light is uniform in all directions (Rayleigh scattering); for larger particles (above approximately 250 nm), the intensity is angle dependent (Mie scattering). If the light is coherent and monochromatic, as from a laser, it is possible to observe timedependent fluctuations in the scattered intensity using a suitable detector such as a photomultiplier capable of operating in photon counting mode. These fluctuations arise from the fact that the particles are small enough to undergo random thermal (Brownian) motion and the distance between them is therefore constantly varying. Constructive and destructive interference of light scattered by neighboring particles within the illuminated zone gives rise to the intensity fluctuation can therefore yield the diffusion coefficient of the particles from which, via the Stokes-Einstein equation, knowing the viscosity of the medium, the hydrodynamic radius or diameter of the particles can be calculated. The time dependence of the intensity fluctuation is most commonly analysed using a digital correlator. This device determines the intensity autocorrelation function which can be described as the ensemble average of the product of the signal with a delayed version of itself as a function of the delay time. The signal in this case is the number of photons counted in one sampling interval. At short delay times, correlation is high and, over time as particles diffuse, correlation diminishes to zero and the exponential decay of the correlation function is characteristic of the diffusion coefficient of the particles. Data are typically collected over a delay range of 100 ns to several seconds depending upon the particle size and viscosity of the medium. Analysis of the autocorrelation function in terms of particle size distribution is done by numerically fitting the data with calculations based on assumed distributions. A truly monodisperse sample would give rise to a single exponential decay to which fitting a calculated particle size distribution is relatively straightforward. The autocorrelation function decays exponentially as follows: 34 Chapter 2: Methods 𝑔(𝜏𝑐)=exp (−𝜏𝑐𝐷𝑠𝑞2) (2.13) where 𝜏𝑐 is termed the correlation delay time, 𝐷𝑠 is the self-diffusion coefficient of the particle, and 𝑞 is the scattering vector defined as the vector difference between the wave propagation vector of the incident and the scattered beam, both of length 2𝜋 𝜆 ⁄: 𝑞=4𝜋 𝜆sin �𝜃 2� (2.14) where 𝜆 is the wavelength of the scattered radiation in the medium. To give the hydrodynamic radius of the particle the Stokes-Einstein relationship is used: 𝑅ℎ=𝑘𝐵𝑇 6𝜋𝜂0𝐷𝑠 (2.15) where 𝜂0 is the viscosity of the continuous phase. In practice, polydisperse samples give rise to a series of exponentials and several quite complex schemes are devised for the fitting process. 2.10 Differential Scanning Calorimeter (DSC) Differential scanning calorimeter (DSC) is an experimental techniques widely used in the study of binary and multicomponent systems containing substances of different classes, as a salt (surfactants), metals, oxides, organic compounds, liquid crystals, etc. including methods available to the biophysical chemist for the study of macromolecules conformation and thermal transitions might be affected by ligand binding. Thermal transition (melting) temperatures (for samples in solution, solid or mixed phases, e.g. suspensions) and enthalpies Chapter 2: Methods 35 are often determined and used to draw the limits of existence of the different phases. A phase transition in a two-component system is often characterized by a single transition temperature and a transition enthalpy, which is done by mechanical analogy with a transition in pure individual substance[13-16]. The advantages of calorimetric techniques arise because they are based on direct measurements of intrinsic thermal properties of the samples and usually non-invasive and require no chemical modifications or extrinsic probes. Furthermore, with careful analysis and interpretation, calorimetric experiments can directly provide fundamental thermodynamic information about the processes involved. In a DSC experiment a sample is heated at constant rate in the calorimeter cell alongside and identical reference cell containing the blank. Differences in heat energy uptake between the sample and reference cells required to maintain equal temperature, correspond to differences in apparent heat capacity, and it is these differences in heat capacity that give direct information about the energetic of thermally induced processes in the sample. From DSC experiment is obtained a thermogram showing the excess heat capacity (Cp sample minus reference) as a function of temperature. For the case of simple globular proteins the thermogram comprises three regions: pre-transition baseline, the endothermic unfolding transition, and post-transition baseline (Fig.2.1). Figure 2.1: Typical thermogram of DSC measurements. The heat capacity (or specific heat) of any substance reflects the ability of the substance to absorb heat energy without increase in temperature, and this central to DSC measurements 36 Chapter 2: Methods and to the fundamental underlying thermodynamics. The absolute enthalpies (H) and entropies (S) may be derived by the heat capacity, (fundamental properties from which all thermodynamic quantities are derived). Changes in enthalpy and entropy ( ∆ H and ∆ S) as a system changes from one state to another (𝐴 → 𝐵) at constant temperature follow directly from the integral definitions: Δ𝐻 =𝐻𝐵−𝐻𝐴=∫ΔCp 𝑇 0𝑑𝑇+Δ𝐻(0) (2.16) Δ𝑆 =𝑆𝐵−𝑆𝐴=∫(Δ𝐶𝑝T) dT ⁄ 𝑇 0 (2.17) where Δ𝐶𝑝=𝐶𝑝,𝐵−𝐶𝑝,𝐴 is the heat capacity difference between states A and B at given temperature. Δ𝐻(0) is the ground state enthalpy difference between A and B at absolute zero. The Calorimeter enthalpy ( ∆ Hcal) is the total integrated area under the thermogram peak which, after appropriate baseline correction, represents the total heat energy uptake by the sample undergoing the transition. The van’t Hoff enthalpy ( ∆ HVH) is an independent estimate of the enthalpy of the transition, based on an assumed model for the process. Here one simply uses the area under the Cp peak at any temperature, divided by the total area, as a measure of the fraction or extent of unfolding that has occurred at that temperature. The relationship between ∆ Hcal and ∆ HVH can sometimes provide insights not accessible from ∆ Hcal alone, for example, if a protein is composed of two identical domains which unfold independently with the same Tm and ∆ Hcal , the ratio ∆ Hcal/ ∆ HVH will be 2.0, while it would be 1.0 if the protein had only a single domain. If, on the other hand, the protein dimerized and the dimer underwent only a single coupled transition then ∆ Hcal/ ∆ HVH ratio will be 0.5. It is clear from this that the calorimetric heat ∆ Hcal refers to heat change per mole while ∆ HVH is heat change per unfolding unit (called cooperative unit). Thus the ratio ∆ Hcal/ ∆ HVH can, in simple cases, be thought of as the number of cooperative units per mole[17]. 2.11 Electron Microscopy techniques Electron microscopy is an important viewing technique to examine biological materials (such Chapter 2: Methods 37 as organisms and cells), a variety of large molecules, medical biopsy samples, metals and crystalline structures, and the characteristics of various surfaces. It can be defined as a specialized field of science that employs the electron microscope as a tool and uses a beam of electrons to form an image of a specimen. Electron microscopy is operated in the vacuum and focuses the electron beam and magnifies images with help of electromagnetic lenses. It has much higher magnification or resolving power than normal light microscope, due to increasing the velocity of electrons results in a shorter wavelength and increased resolving power. The two basic types of electron microscopes are the scanning electron microscope and the transmission electron microscope. The relatively high magnification range of both basic types of electron microscopy allows investigators to detect in specimens much greater detail than those examined by light microscopy.[18] The Transmission Electron Microscopy (TEM) projects electrons through an ultrathin slice of the specimen and produces a two dimensional image. The sample is bombarded by a highly focused beam of single-energy electrons. The electrons are accelerated by an electric potential and focused by electromagnetic lenses onto the sample. The beam has enough energy for the electrons to be transmitted through the sample. The transmitted beam contains information about electron density, phase and periodicity; this beam is used to form an image. The scanning electron microscope reveals information about the sample, for example, external morphology, chemical composition, and crystalline structure and orientation of materials making up the sample. The electrons interact with the sample within a few nanometers to several microns of the surface, depending on beam parameters and sample type. Electrons are emitted from the sample primarily as either backscattered electrons or secondary electrons. Secondary electrons are the most common signal used for investigations of surface morphology, which provides one of the main signals for image formation in the scanning electron microscopy. Electrons of low energy (up to 50 eV) may be ejected from the specimen; these are secondary electrons. Once these electrons escape from the sample surface, they are typically detected by a photomultiplier detector. The SEM image formed is the result of the intensity of the secondary electron emission from the sample at each x,y data point during the rastering of the electron beam across the surface Some energy from the beam electrons is transferred to the conduction band electrons in the sample, providing enough energy for their scape from the sample surface as secondary electrons. Since these electrons travel farther into the sample than the secondary electrons, they can emerge from the sample at a much larger distance away from the impact of the incident beam which makes their spatial distribution larger. 38 Chapter 2: Methods This occurs in a vacuum environment ranging from 10-4 to 10-10 Torr. The electrons are guides to the sample by a series of electromagnetic lenses in the electron column. The resolution and depth of field of the image are determined by the beam current and the final spot size, which are adjusted with one or more condenser lenses and the final, probe-forming objective lenses. The lenses are also used to shape the beam to minimize the effects of spherical aberration, chromatic aberration, diffraction, and stigmatism. Figure 2.2: Scheme of Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM). 2.12 Atomic Force Microscopy (AFM) The AFM is a powerful new tool that consists of scanning a sharp tip on the end of a flexible cantilever across a sample surface while maintaining a small, constant force. The main difference between these types of microscopy and the AFM is, as the name suggests, interactive forces between the sample and the tip. The force most commonly associates with atomic force microscopy is an interatomic force called the van der Waals force. This technique has the advantage of imaging almost any type of surface, including surfactants, polymers, ceramics, and biological samples either resolution at an atomic scale. AFM technique mainly uses a laser beam deflection system, where a beam is reflected from the back of the reflective AFM lever and onto a position-sensitive detector. The tips typically have an end radius of 2 nm to 20 nm, depending on tip type and are microfabricated from Si or Si3N4. Chapter 2: Methods 39 It is known that the AFM technique is based on the forces between the tip and sample, but this force is not measured directly. The force is calculated by maintaining a constant cantilever deflection; the force between the tip and the sample remains constant, and is calculated from Hook’s law, which is given by: 𝐹=−𝑘𝑥 (2.18) where 𝐹 is the force, 𝑘 is the spring constant, and x is the cantilever deflection. A plot of the laser deflection versus tip position on the sample surface provides the resolution of the hills and valleys that constitute the topography of the surface. A scheme can be seen in Figure 2.3: Figure 2.3: A deflection AFM. The cantilever deflection is detected by a reflected laser beam and photodiode. The tip is integrated with the cantilever. The substrate is moved in the xyz directions by a piezoelectric scanner. A feedback loop controls the rate of z movement. In AFM, different modes of operation are applicable to specific application requirements: Contact mode, Non-Contact mode and Tapping mode. The two most commonly used modes of operation are Contact Mode AFM and Tapping Mode AFM, which are conducted in air or liquid environments. Contact Mode consists of raster-scanning the probe or sample while monitoring the change in cantilever deflection with the split photodiode detector. A feedback loop maintains a constant cantilever deflection by vertically moving the scanner at each (x,y) data point is stored by the computer to form the topographic image of the sample surface. This feedback loop maintains a constant force during imaging. 40 Chapter 2: Methods Tapping Mode depends on the detection of amplitude of a driven cantilever oscillation; the cantilever is driven to oscillate close to its resonance frequency (~300 kHz) and lightly “tapping” the tip on the surface during the scanning. The laser reflection method is used to detect the root-mean-square (RMS) amplitude of the cantilever oscillation. A feedback loop maintains constant amplitude by moving the scanner vertically at every (x,y) data point. By maintaining constant oscillation amplitude a constant tip-sample interaction is maintained during imagining. The advantage of Tapping Mode over contact mode is that it eliminates the lateral, shear forces present in contact mode. This enables Tapping Mode to image soft, fragile, and adhesive surfaces without damaging them, which can be a drawback of contact mode AFM. Chapter 2: Methods 41 An increase in the temperature appears to cause a small decrease in the aggregation number in aqueous medium of ionics, presumably because 𝑎0 is increased due to thermal agitation. If small amounts of hydrocarbons or long-chain polar compounds are added to an aqueous solution of a surfactant above its cmc, these normally water-insoluble materials may be solubilized in the micelles. This solubilization generally causes an increase in the aggregation number of the micelle, and as the amount of material solubilized by the micelle increases, the aggregation continues to increase until the solubilization limit is reached. 3.1.2 Factors affecting the critical micellar concentration Among of factors known affect the cmc in aqueous solution, such as the structure of surfactants, the presence of added electrolyte in solution, the presence in solution of various organic compounds, the presence of a second liquid phase, and the temperature of the solution. Among of factors which affect the cmc in aqueous solution, the structure of surfactants, the presence of added electrolyte in solution, the presence in solution of various organic compounds, the presence of a second liquid phase, and the temperature of the solution have the greater impact on it. In general, the cmc in aqueous media decreases as the hydrophobic character of the surfactant increases. 3.1.3 The Hydrophobic Group An increase in the length of the hydrocarbon chain in the unbranched portion of the chain decreases the cmc. Generally, used rule for ionic surfactants is that the cmc is halved by the addition of one methylene group to a straight-chain hydrophobic group attached to a single terminal hydrophilic group. When the hydrophobic group is branched, the carbon atomos on the branches appear to have about one-half the effect of carbon atoms on a straight chain. When carbon-carbon double bonds are present in the hydrophobic chain, the cmc is generally higher than that of the corresponding saturated compound, with the cis isomer generally having a higher cmc than the trans isomer. Similarly, introduction of polar groups such as –Oor –OHinto the chain normally increases the cmc in aqueous medium at room temperature. 48 Chapter 3: Theoretical Aspects 3.1.4 Hydrophilic Group In aqueous medium, ionic surfactant have much higher cmc than nonionic surfactants containing equivalent hydrophobic groups. This is due to electrostatic repulsion between the head groups of the neighboring surfactant monomers within the micelles. Zwitterionics appear to have slightly smaller cmc than ionics with the same number of carbon atoms in the hydrophobic group. Surfactants containing more than one hydrophilic group in the molecule show larger cmc than those with one hydrophilic group and the equivalent hydrophobic group. For a given chain length, variation of the actual ionic headgroup present on the surfactant can markedly affect the cmc. The cmc in alkyl ionic surfactants follows the order: aminium salts > carboxylates > sulfonates > sulfates[23]. 3.1.5 Effect of Counterions The critical micellar concentration in aqueous solution reflects the degree of binding of the counterions to the micelle. Increased binding of the counterion, in aqueous systems, causes a decrease in the cmc of the surfactant. The extent of adsorption of counterios depends also on the properties of the counterions. For example, an increase in the valency, as well as in the polarizability of the counterions, decreases the cmc. An increase in the hydrated radius, on the other hand, increases the cmc. For a given hydrophobic chain and anionic head group, such as dodecylsulfates, the cmc decreases in the order: Li+ > Na+ > K+ > Cs+ > Mg2+ > Ca2+. For cationic series, dodecyltrimethyl ammonium, for example, the cmc decreases as: F- > Cl- > Br- > I-[19]. 3.1.6 Effect of additives The electrolyte effect is more pronounced for anionic and cationic than zwitterionic surfactants and more pronounced for zwitterionics than nonionics. This effect consist of in aqueous solution the presence of electrolyte causes a decrease in the cmc of most surfactants. The dependence of cmc on electrolyte level can be described using the following equation: Chapter 3: Theoretical Aspects 49 log 𝑐𝑚𝑐 =−𝐴log 𝐶𝑒+𝐵 (3.1.4) where 𝐴 and 𝐵 are constants and 𝐶𝑒 is the total concentration of the counterions. Zwitterionics and nonionics exhibit less dependence on electrolyte levels than ionics surfactants, and the corresponding relation is given by: log 𝑐𝑚𝑐 =−𝐾𝐶𝑠+𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 (3.1.5) where 𝐾 is a constant for a particular surfactant-electrolyte system at a given temperature and 𝐶𝑠 is the salt concentration. Addition of an oppositely charged surfactant in small amounts to a given ionic surfactant can also result in a significant decrease in the cmc[24]. Note, however, that addition of equimolar amounts of oppositely charged surfactants can result in precipitation and the region of homogeneus solution is restricted to very low total concentrations. 3.1.7 Temperature effect The process of micellization itself is dependent on temperature in a complex way. Ionics surfactants typically exhibit a minimum in cmc with increase in temperature. This behavior is a consequence of the decrease in hydrophobicity of the surfactant molecules, an initial decrease in the cmc with the temperature. The increase in temperature causes a decrease in the hydration of the hydrophilic head group, favoring micelles formation. At the same time the increase in temperature causes a breakdown in the structured water molecules surrounding the hydrophobic alkyl chains, disfavoring the micellization process and the cmc increasing with temperature. The U-shaped curve observed is the outcome of the interplay of these two effects and was observed for different ionic and nonionic surfactants[25]. The effect of counterions is even more relevant on the minimum due to their different hydration capabilities. It was postulated that the cmc of ionic surfactants should follow the Hofmeinster series[26,27]. 50 Chapter 3: Theoretical Aspects List of papers included in this section • On the Self-Assembly of a Highly Selective Benzothiazole-Based TIM Inhibitor in Aqueous Solution. • Self-assembling drugs: A new therapeutic strategy. • Hydrogenated/fluorinated catanionic surfactants as potential templates for nanostructure design. Chapter 3: Theoretical Aspects 51 3.2 Protein-Ligand Interactions 3.2.1 Introduction For decades, researches focused their attention on functions and characteristics of proteins and protein surfactants mixtures based on their widespread applications (purification of receptors in their active forms, solubilization of enzymes lipoprotein, SDS-PAGE, isolation of hydrophobic proteins, drug delivery, or cosmetic industry). The recent flurry of research activity in biomedical, bioelectronics, or molecular templating of these system claims for substantial progress in determining the physical principles that underlie these phenomena. Proteins are structures with a great importance. Many medical, health and biological projects and advances are concerned with these materials. The stability and functionality of proteins are strongly dependent on the medium properties. 3.2.2 Protein structure Proteins are linear sequences of amino acids linked together by peptide bonds. In solutions contain a mixture of different types of chemical groups, such as non-polar, polar and electrical charged. For this reason it is possible to characterize a typical protein as an amphoteric molecule and it is not surprising that most small amphiphilic molecules will interact strongly with proteins. 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 (Fig.3.2A) 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 α-amino group of one amino acid and the αcarboxyl 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 52 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 α-helix (Fig.3.2B) and the β-sheet (Fig.3.2C). In the rod-like α-helix, the amino acids arrange themselves in a regular helical conformation. The β-sheet structure is a pleated sheet composed of β-strands in parallel or antiparallel arrangement depending on whether they run in the same direction or in opposite directions, respectively. β-pleated sheets are always slightly curved and, if several polypeptides are involved, the sheet can close up to form a β-barrel. Regions of the polypeptide chain that are not in a regular secondary structure are said to have a coil or loop conformation. The secondary structure elements fold into structural units, called domains, which comprise the tertiary structure. Figure 3.2: Protein structure (A) primary structure. Secondary structure: (B) α-helix (C) β-sheets. (D) Tertiary structure. (E) Quaternary structure. Chapter 3: Theoretical Aspects 53 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 (Fig.3.2D). 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 (Fig.3.2E). 3.2.3 Protein-Ligand interactions 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. As mentioned above, proteins are formed by a mixture of amino acids in unique combinations. The different nonpolar, polar and charged properties of the amino acids lead to complex interactions that determine the proteins’ secondary, tertiary and quaternary structure. Due to the hydrophobic and hydrophilic properties of amino acids, a protein exhibits a dualism that makes small amphiphilic molecules interact with proteins. The native structure is the three-dimensional arrangement of the functional protein when it is located in its natural environment. Changes in the environment, e.g., a rise in temperature, variation of pH or addition of denaturants, may cause denaturation of the protein. When a protein is denaturated, the characteristic three-dimensional arrangement is disrupted (the protein unfolds), and, consequently its biological function is lost. 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)[28]. 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[29]. 54 Chapter 3: Theoretical Aspects Much of biotechnological, medical and drug delivery systems aims to predict their behavior on the basis of the set of molecules involved, mainly proteins and drugs. Understanding the interactions between these molecules is therefore crucial to such efforts. Although many thousands of interactions are known, precise molecular details are available for only a tiny fraction of them. The difficulties that are involved in experimentally determining atomic structure for interacting proteins make predictive methods essential for progress. Structural details can ultimately turn abstract system representations into models that more accurately reflect biological reality[5]. Understanding protein-ligand interaction is central, for example, to drug design and discovery of new medicines to benefit human health, and as well as, stabilizers functions in natural systems like in processed food used to stabilize emulsions, and as stabilizers for inorganic particles in paints or photographic films. A high level of steric complementary between the protein and the ligand is described by the lock-and-key paradigm and a high degree of complementary of hydrophobic and polar parts of both entities, namely, the binding site of the protein and the ligand. There is usually high complementary of the surface properties between the protein and the ligand. Lipophilic parts of the ligands are most frequently found to be in contact with lipophilic part of the protein. Polar groups are usually paired with suitable polar protein groups to form hydrogen bonds or ionic interactions. The experimentally determined hydrogen bond geometries display a fairly small scatter-in other words, the hydrogen bond geometry is strongly preserved. With very few exceptions, there are not repulsive interactions between the ligand and the protein. Direct interactions between the protein and the ligand are very important for binding, and these binds in an energetically favorable conformation[30]. The most important direct interactions are hydrogen bonds, ionic interactions, hydrophobic interactions, cation-π interactions and metal complexation. Therefore, in the ligand design process one has to ensure that polar functional groups, either of the protein or the ligand, will find suitable counterparts if they become buried upon ligand binding. 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 Chapter 3: Theoretical Aspects 55 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 (Fig. 3.3). 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[31]. Compared ionics, nonionic surfactants bind weakly to the proteins due to the absence of electrostatic interactions, thus making micelle formation in bulk more favourable. 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[19]. 56 Chapter 3: Theoretical Aspects Figure 3.3: Typical non-bonded interactions found in protein-ligand complexes. However, this short characterization is only a part of the story: ligand and binding site flexibility, distortion energies, desolvation effects, entropy, molecular electrostatic field complementary, and other effects are often qually important. Thus, the interpretations of data in the extensive literature on the unfolding of proteins in aqueous solution by amphiphilic molecules (drugs) have been recently analyzed [32,33]. During the 2000s improved methods were revised for improving assumptions associated with incomplete equilibrium constants, contradict model-free interpretation of the data and misuse of the Van’t Hoff relation is really common. In addition, the notion that proteins are simply three-dimensional, compact objects has been questioned: while proteins need to keep their specific Chapter 3: Theoretical Aspects 57 List of papers including in this section • Assessment of interactions between four proteins and benzothiazole derivatives by DSC and CD. • Mechanisms of fibrinogen-acebutolol Interactions: Insights from DSC; CD and LS. • Surface characterization and AFM imaging of mixed fibrinogen-surfactant films. • Fibrinogen stability under surfactant interaction • Investigating the effect of an arterial hypertension drug on the structural properties of plasma protein 64 Chapter 3: Theoretical Aspects 3.3 Hydrogels 3.3.1 Introduction Hydrogels are cross-linked 3-D networks containing covalent bonds (produced by the reaction of one or more comonomers), physical cross-links (due to chain entanglements), hydrogen bonds, strong van der Waals interactions, and crystallite associations (bringing two or more macromolecular chains) [39]. These structures have been recently attracted much interest in the biomaterials sector because of their ability to entrap large quantities of water or biological fluids. This high water content mimics the natural environment, which gives them excellent biocompatibility while the three-dimensional network provides mechanical support. Recently the ability of proteins and peptides to self-assemble into ordered supramolecular architecture on the meso to macroscopic length scales has attracted considerable attention in the development of novel biomaterials due to their potential biocompatibility and biodegradability. Consequently they have found a wide range of applications in the medical, pharmaceutical and biomaterials sectors (drug delivery, materials for artificial organs) [40-42]. The hydrogels properties can be modulated by varying the synthetic factors, such as vessel, reaction time, reaction temperature, monomer type, type of crosslinker, monomer concentration, and type and amount of initiator. Based on different aspects of hydrogels their classification may be based on the source: • Natural, synthetic gels or hybrid hydrogels: composed of synthetic and natural molecules. • Nature of the crosslinking: covalent or physical gels • Nature of the network: homopolymer networks, copolymer networks, interpenetrating networks or double networks. • Physical structure: homogeneous (optically transparent) microporous, and macroporous hydrogels • On their fate in an organism: degradable and nondegradable hydrogels. There are three classes of molecules that can be used to produce hydrogels: polymers, peptides and proteins. Polymers have been used extensively to create hydrogels; however many are not adhesive to cells. Consequently, recent efforts have focused on modifying the material to induce biocompatibility for tissue engineering application [43-46]. 65 3.3.2 Hydrogels Structure Based on the nature of the crosslinking force, hydrogels can be categorized as chemical (covalent) or physical gels. In chemical hydrogels the network of chemical links joining different chains can be obtained by cross-linking bulk polymers or polymers in solution. Due to the covalent crosslinks, chemically crosslinked hydrogels generally have high mechanical strength. On the other hand, the chemical reactions required to form crosslinks might potentially affect the structure and biological activity of encapsulated pharmaceuticals. Moreover, chemical crosslinking often requires crosslinking reagents or catalysts that are toxic towards cells [47-48]. Physical hydrogels chains are held together by non-permanent interactions, such as ionic interactions, hydrophobic interactions, hydrogen bonds and specific biomimetic interactions [49-52] (Figure 3.3.1). These interactions can be disrupted by changes in the environment, such as temperature, pH, ionic strength, presence of specific solutes, and stress; consequently, the formation of physical hydrogels may be reversible. Figure 3.3.1: Schematic representation of: A) Chemical and B) Physical crosslinked hydrogels. The properties of both chemically and physically crosslinked hydrogels can often be controlled by a wide variety of parameters, such as the number of crosslinks, the chemical structure of the polymer main chain and the water content [53]. In both cases, the density of cross-links is crucial in determining the properties and applications of the gels, as it is responsible for the swelling behavior and therefore for the combine solid-like and liquid-like characteristics [54]. The swelling capacity of a hydrogels can be determined by the amount of space inside the hydrogels network available to accommodate water. Hydrogels can also be classified as 66 Chapter 3: Theoretical Aspects neutral, anionic, or cationic depending on the charges of the building blocks (Fig. 3.3.2). Nonionic hydrogels swell in aqueous medium solely due to water-polymer interaction. Cationic and anionic hydrogels swelling are dependent on the pH of the aqueous medium. At low pH cationic hydrogels display superior swelling due to their chain dissociation; the same for anionic hydrogels, at higher pH dissociate more, and hence exhibit superior swelling in neutral to basic solutions. Figure 3.3.2: Different Hydrogels structures. Interestingly, a large number of biopolymers possess property to self-structure upon temperature variation. Two different types of temperature sensitive materials can be distinguished: upper critical solution temperature and lower critical solution temperature materials. In the first case, the gelation of many biopolymers is induced by the reversible temperature-sensitive formation of intermolecular hydrogen bonds. The second class, at low temperatures, a homogeneous solution is obtained. Upon heating, aggregation of the hydrophobic groups occurs, inducing phase separation and hydrogel formation. The endothermal gelation is driven by an entropy change. In contrast with the increase in order during the aggregation of the hydrophobic segments, the entropy increase during the hydrogel formation. This is due to the large amount of water molecules released by the hydrophobic part of the polymer[55]. Gelation thus occurs spontaneously upon heating because the entropy (𝑇Δ𝑆) compensates for the unfavorable enthalpy (Δ𝐻). For example, the gelation of methylcellulose (MC) is mainly induced by intermolecular, hydrophobic interactions. Consequently, these macromolecules are fully hydrated at low temperatures. Upon heating, gradual dehydration occurs, resulting in a viscosity increase. Near the transition temperature, polymer-polymer interactions are dominant and result in the formation of a polymer network[56]. It was demonstrated that MC is a promising candidate material to be applied as brain cell support, including the treatment of multiple-site injuries, Chapter 3: Theoretical Aspects 67 and in combination with hydroxyapatite, cellulose shows potential to be applied for bone tissue engineering [57]. Protein gels that form in-vivo are of profound physiological importance. The human body is approximately 65% water by mass and much of that water is contained in tissues throughout the body [58]. For example, even though dermal layers are approximately 70% water, skin is flexible enough to permit a large range of motions and also elastic enough to support the underlying tissues and fluids [59]. Another important example is the blood clot, which, despite its high water content, is strong enough to support haemostasis at an injury site while also facilitating tissue repair [60]. Both of these tissues have an underlying microscopic network of interconnected protein fibers that form through the self-assembly of collagen and fibrin respectively Proteins are essentially polymers of amino acids and are known to form β-sheets-rich fibrils (nanometers in width and micrometer in length) that also further self-organize and entangle to form three-dimensional hydrogels under appropriate conditions. It is through that the fibrillar network is stabilized by intermolecular and/or intramolecular hydrogen bonding, electrostatic interactions and hydrophobics effects. A typical example of a protein motif used in synthesis of hybrid hydrogels or protein-based hydrogels is the coiled-coil. Coiled-coils have been found in over two hundred native proteins [61]. Structurally the coiled-coil is a supercoil formed by two or more strands of α-helices, will be used as an example to demonstrate the potential for the design of well-organized hydrogel structures. The primary sequence of a typical coiled-coil is composed of 7-residues repeats, designed as heptads (Figure 3.3.3, left panel). The amino acid residues in a heptad are conventionally denoted as “a, b, c, d, e, f, g”. Hydrophobic residues at positions “a” and “d” form an inter-helical hydrophobic core, providing a stabilizing interface between the helices. Charged residues at positions “e” and “g” form electrostatic interactions, which contribute to coiled-coil stability and mediate specific association among helices. 68 Chapter 3: Theoretical Aspects Figure 3.3.3: Left panel: helical wheel diagram of a two stranded coiled-coil. Right panel: βsheets structure motif. β-sheets are important structural elements in proteins. β-strands are aligned adjacent to each other and are stabilized by hydrogen bonds between the carbonyl oxygen of an amino acid in one strand and the backbone nitrogen of a second amino acid in another strand (Figure 3.3.3, right panel). The strands (at least two, but frequently more) can arrange in parallel or antiparallel fashion to form the β-sheets. 3.3.3 Hydrogels Characterization Hydrogels can be described in a rheological way. Rheology is the science of flow and deformation of matter, deformation (shear strains and shear rate) that occurs when a material is subjected to a stress (force). The relationship between stress and deformation is a property of the material. Therefore, it can be define “Rheology” as the study of stress-deformation relationship. The stress (force per unit area) can be applied in various ways: as a compression, as a tension, or as a shearing process. In compression and tension, dilute dispersions behave very much like simple liquids, especially if the particles are rigid and/or incomprenssible. Only in highly concentrated dispersions does one encounter unusual behavior under tension, while under compression most condensed materials (solid or liquid) behave rather similarly Chapter 3: Theoretical Aspects 69 Strain, 𝛾=𝑥(𝑡) 𝑦0 Strain rate, 𝛾󰇗 =𝑉 𝑦0 𝑑 𝑥(𝑡) 𝑑 𝑡 Viscosity, 𝜂=𝜏 𝛾󰇗 Figure 3.3.4: Scheme of application of a shearing stress to a material, produces a strain, 𝛾=tan 𝜃 3.3.4 Theorical aspects Dynamics tests allow analyze the viscoelastic response of a material. These tests, also called “oscillatory test”, consists on to apply a shear stress or shear strain that is varied sinusoidal over time while is during the test. Oscillatory or dynamic test consists basically undergo to the material to a periodic deformation, which usually it is a simple sinusoidal shear that could be generated moving forward and backward to the top plate, consisting of deforming a material positioned between two parallel plates to move linearly the upper plate at given distance. 3.3.5 Response to an oscillating shear field The shear strain applied to the material can be expressed as a function of time according to: 70 Chapter 3: Theoretical Aspects 𝛾=𝛾0sin 𝜔𝑡 (3.3.1) where 𝛾 is the shear strain, 𝛾0 is the maximum amplitude of the strain corresponding to simple harmonic motion, 𝜔 is the frequency and 𝑡 is the time applied in the test. The shear rate is also a periodic function: 𝛾󰇗 =(𝑑𝛾) (𝑑𝑡)=𝛾0𝜔cos(𝜔𝑡) = 𝛾󰇗0cos(𝜔𝑡) (3.3.2) A material subjected to small sinusoidal strain, that guarantees a linear viscoelastic answer, responds after a certain number of cycles with a shear stress that also follows a sinusoidal function with respect to the time at the same frequency but with an amplitude characteristic of the nature of such material. Hook’s law describes ideal mechanical behavior using a constitutive equation in which stress and strain are related through a constant. If the material behaves as an ideal solid, the response is purely elastic and therefore the shear strain depends linearly as: 𝜎=𝐺𝛾 (3.3.3) where 𝐺 is the ridigity modulus, and therefore the shear strain waves and shear stress will be in phase: 𝜎=𝐺𝛾0sin 𝜔𝑡 (3.3.4) Newton formulated a similar proposal concerning properties of liquids. If the material was a viscous ideal fluid, it will fulfill the Newton’s law: 𝜎=𝜂[(𝑑𝛾) (𝑑𝑡)⁄] (3.3.5) Chapter 3: Theoretical Aspects 71 This law assumes that a force (or resistance) is proportional to a velocity (of movement). The coefficient of proportionality, 𝜂, is called viscosity (or coefficient of viscosity) And taking into account the expression (3.3.2), the wave corresponding to the shear stress would be out of phase in 90o with respect to shear strain wave: 𝜎=𝜂𝛾󰇗0cos(𝜔𝑡)=𝜎0cos(𝜔𝑡) = 𝜎0sin(𝜔𝑡+90) (3.3.6) Both models (Hook and Newton models) represent properties of many materials and work well describing their behavior with considerably high degree of accuracy. However, there are numerous other real materials which are not described by above mentioned Hook and Newton laws. Rheology relies on the idea that non-Newtonian and non-Hookean materials exist in reality. Such materials are said to be visco-elastic and they may be intrinsically solids or liquids, depending on which of the two characteristics is dominant. A viscoelastic material shows an intermediate behavior between pure viscous and pure elastic ones, hence the shear stress displays a phase angle between 0o and 90o respect to the shear strain (Fig.3.3.5). The shear stress can be expressed mathematically as: 𝜎=𝜎0sin(𝜔𝑡+𝛿) (3.3.7) where 𝜎0 is the maximum amplitude corresponding to the shear stress and 𝛿 is the phase angle related to the deformation. 72 Chapter 3: Theoretical Aspects Fig.3.3.5: Elastic solid, viscous liquid and viscoelastic behavior of hydrogels. If the expression (3.3.7) is developed trigonometrically we have: 𝜎=𝜎0(sin 𝜔𝑡cos 𝛿+ sin 𝛿cos 𝜔𝑡) (3.3.8) An analysis of the expression (3.3.8) suggests that the wave related to the shear stress can be decomposed in two waves of the same frequency, a phase corresponding to the shear strain wave (sin 𝜔𝑡) and another one with a phase angle of 90o (cos 𝜔𝑡). Thus, (3.3.8) can be displays as follows: 𝜎=𝜎′+𝜎′′ =𝜎′0 sin 𝜔𝑡+𝜎′′0cos 𝜔𝑡 (3.3.9) From the comparison between (8) and (9) it can deduce that: Chapter 3: Theoretical Aspects 73 Chapter 4: RESULTS AND DISCUSSION 4. Results and Discussions In this chapter we expose and discuss the obtained results. Early results have been published, we include the corresponding articles. Other studies, with both quantitative and qualitative results, have been done recently but the results have not yet been published so we include the data and analysis in the present form. 4.1 Published articles 4.1.1 Relevant Aspects of published articles. • Self-Assembly of Surfactants I. On the self-assembly of a highly selective benzothiazole-based TIM inhibitor in aqueous solution. Benzothiazole derivates are selective and high affinity inhibitors of different triosephosphate isomerases (TIM) including Tripanozome cruzi (Tc) and Tripanosome brucei (Tb) TIMs from Trypanosoma cruzi (TcTIM), the parasite that causes Chagas disease that affects 18 million people in American Continent. Effective treatments for trypanosomiasis are still challenge with one of the benzothiazole derivate, 3-(2Benzothiazolylthio)-propanesulfonic acid (BTS). It has been shown that premature aggregation of amphiphilic molecules below the corresponding critical aggregation concentration induced by presence of macromolecules is possible. These findings, together with our own interest in drug self-assembly, drug-protein interaction and even TcTIM (the actual target of BTS), encouraged us to perform this comprehensive study of BTS self-assembly as a function of its concentration, temperature and also salt concentration. 83 II. Hydrogenated/fluorinated catanionic surfactants as potential templates for nanostructure design. Catanionic surfactants are chemical species consisting of an amphiphilic anionic and an amphiphilic cationic molecule. Mixtures of heterogeneous molecules which self-assemble in aqueous solution represent an interesting alternative for the design of nanoparticles due to its higher flexibility when compared to systems based on homogeneous molecules. A combined experimental/computational study involving density, sound velocity, and dynamic light scattering measurements, together with transmission and scanning electron, as well as confocal laser microscopy images, and molecular dynamics simulations of equimolar mixtures of these two surfactants at several concentrations and at room temperature was performed for this aim. III. Self Assembly drugs: A new therapeutic strategy. Here we define a new strategy namely “self assembled drugs” as a self assembled structure (spherical or worm-like micelles, vesicles or liquid crystals) that is formed uniquely by drugs that combined assemble in such structures. All the individual compounds should display a therapeutic effect by themselves and resulting selfassembled structure display at the same time the role of support vector. Many drugs display amphiphilic properties thus they can be combined with hydrotropic drugs to form different kind of self-assembled structure, “self-assembled drugs”. By combination of hexadecyltrimethylammonium bromide (CTAB) and dicloxacillin (DC) resulting in the formation of worm-like micelles. Both drugs display well known therapeutic activity. The experimental techniques and theoretical background are now well established making them easy to characterize under different conditions. In this case we investigated this system using density and sound velocity, dynamic light scattering and cryo-electron microscopy (Cryo-TEM) and UV. We expect our work to encourage the scientific community to investigate new selfassebled drugs and their advantages over more conventional strategies in drug delivery. 84 Chapter 4: Results and Discussion • Protein-Ligand Interactions IV. Assessment of Interaction between four proteins and benzothiazole derivates by DSC and CD. The interaction of small molecules with macromolecules of biological systems and with specific receptors sites on supramolecular organizations is one of the most extensively studied phenomena in biophysical research. Benzothiazoles have been reported to possess potent anticancer properties due to their structural similarity with naturally occurring purines as they can easily interact with biomolecules of the living systems. The interaction of this drug with different proteins is attractive due to possible conformational changes or unfolding transitions induced by BTS. In order to understand the thermal stability of the lysozyme, ovalbumin, myoglobin and fibrinogen and the effect of BTS concentration we have carried out differential scanning calorimetric studies. Circular dichroism (CD) spectroscopy has been also employed to determine the effect of BTS on the secondary structure content. Measurements by DSC have yielded information regarding transition temperature (Tm), calorimetric enthalpy (∆H), van’t Hoff enthalpy (∆HV). V. Mechanism of fibrinogen-acebutolol interactions: Insights from DSC, CD and LS. Many pharmacologically active compounds are amphiphilic molecules and many of these drugs exhibit the same behavior as traditional surfactants, i.e., they tend to selfassociate to form micelles usually with a small aggregation number. This phenomenon is very important for their application, particularly for their effects upon membrans or pharmaceutical formulations. Previously have been investigated the complexation of different proteins with amphiphilic ligands (surfactants, lipids and drugs) using physicochemical methods. From these results is possible to obtain a thermodynamic picture of the nature of the protein-amphiphilic interaction. A very important prescribed drug for hypertension treatment is acebutolol, a beta-selective betablocker that exhibits, as many b-adrenoceptor blocking agents, a range of pharmacological effects which arise as a result of modification of the cell membrane. One of the routes of administration of acebutolol is intravenous, where immediately delivered to the bloodstream to interact with plasma proteins as fibrinogen (who major function is to form fibrin clots). In this paper was investigated the nature of the interactions of acebutolol with fibrinogen using differential scanning calorimetry, dynamic light scattering and circular dichroism. Chapter 4: Results and Discussions 85 VI. Surface Characterization and AFM imaging of mixed fibrinogen-surfactant films. Is well known that the behavior of proteins at interfaces is important to numerous phenomena in biology and biochemistry where adsorption occurs onto biomaterial surfaces. Due to the strong amphipatic nature of proteins, they tend to adsorb at the fluid interfaces, lowering the interfacial tension. In addition also is well known that many small amphiphilic molecules bind strongly to proteins to form protein-surfactant complexes and as a consequent can stabilize the interface through different mechanism. Hydrogenated and fluorinated surfactants can act as stabilizer at low surfactant concentration or as a denaturant of proteins at high surfactant concentration. Details of the specific interactions occurring between proteins and fluorinated surfactants are still unknown. Fibrinogen is considered to be a major inhibitor of lung surfactants’ function at the lining layer of alveoli, and also one of the most relevant protein that adsorb onto biomaterial surface. This article study the adsorption behavior of fibrinogen in presence of hydrogenated and fluorinated surfactant at the air-water interface using surface techniques including surface tension, surface dilatational rheology, and atomic force microscopy (AFM). VII. Investigating the effect of an arterial hypertension drug on the structural properties of plasma protein. Protein-drugs molecule interactions have been extensively studied by a variety of experimental methods. 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 biotechnological applications, such as drug delivery, sensors, and catalysts. Propranolol is a β-adrenergic blocking agent. It is the most prescribed drug in treating hypertension; it is also applied to manage chronic stable angina. Previous studies on β -adrenoceptor blocking agents have shown that their pharmacological effects arise as a result of modification of the cell membrane. Due to the importance of fibrinogen in a several function of the body we have investigated the effect of propranolol with this plasma protein using different experimental techniques such as differential scanning calorimeter (DSC), dynamic light scattering (DLS), circular dichroism (CD), surface tension and atomic force microscopy (AFM). Such characterization may provide crucial information towards the design of drugs with optimal performance. 86 Chapter 4: Results and Discussion VIII. Fibrinogen stability under surfactants interactions. Contemporary studies have been centered on the use of fibrinogen for nonviral vector delivery, scaffolds, and biocompatibility studies. Despite the very interesting applications of this protein, its interaction with small molecules as surfactants has not been exploited yet. We have focused our research on the stability, possible conformational changes and interactions between fibrinogen and hydrogenate/fluorinated surfactants with a view of better understanding the mechanism that are responsible for the adsorption of amphiphile molecules to biopolymers. These surfactants allow us to compare the differences between hydrocarbon and fluorocarbon surfactants with the same chain alkyl chain with those where the hydrocarbon chain is 1.5 times longer than the fluorocarbon chain. We made use of differential scanning calorimetry (to get insight into thermodynamic parameters), absorbance spectroscopy, and circular dichroism, as well as small-angle X-ray scattering (to have information on protein conformational changes). A proposal of this work could be of interest for applications in biomaterial science where devices should be created with improved hemocompatibility. • Hydrogels IX. Rheological properties of ovalbumin hydrogels as affected by surfactants addition. Hydrogels are three-dimensional networks of crosslinked hydrophilic polymers, which are able to retain considerable amounts of water. The ability to form a gel is an important function of proteins in food systems. Most food protein gels are formed by denaturation, aggregation, and gelation during heating process. The aggregation of globular proteins is regulated by many factors such as covalent bonding, electrostatic interactions, hydrogen bonding, hydrophobic interactions, and van der Waals forces. If external conditions are changed, the protein in solution may unfold and expose interior hydrophobic regions and sulfydryl groups to the solvent. The partially denaturated proteins can aggregate and, under appropriate conditions, produce a macroscopically continuous three-dimensional gel network that entraps and restricts the motion of the solvent. Nowadays, interaction of protein with surfactants has been is of great importance in the fields of industrial, specifically interactions of ovalbumin with surfactant has been a hotspot these days. Concerning the interest of the industry in ovalbumin hydrogels range from emulsifying activity (it is higher at acid pH) or drug delivery systems. Chapter 4: Results and Discussions 87 We have investigated the effect of sodium perfluorooctanote, sodium octanoate, and sodium dodecanoate, on the properties and structure of the ovalbumin gels with the purpose of analyze how the presence of fluorine atoms in the alkyl chain affects the gel structure. The physicochemical properties of these samples were evaluated by using rheological measurements that are easy to conduct, especially in high concentration range proteins. X. Mimicking Natural Fibrous Structures of Opals by Means of a MicroemulsionMediated Hydrothermal Method. The synthesis of new materials made of particles, rods and wires with dimensions in the nanoscale is among the most active areas of research in science due to the unique properties of these materials compared to conventional materials made from micron sized particles. Metal oxides including silica represent a large class of inorganic materials that find many scientific and technological applications. For example, siliconbased nanomaterials are of the great interest because of their potential applications in constructing electronic and optoelectronic nanodevices. We have designed a simple and controllable route for the synthesis of opals-CT materials with unusual fibrous microstructures similar to those existed in the nature, using a bottom-up microemulsion droplet system as chemical microreactor. The microcrystalline 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 calcinations temperature. 88 Chapter 4: Results and Discussion