Characterization of nanomaterials, bovine serum albumin and their mixtures using a dynamic surface tension detector (DSTD), dynamic light scattering (DLS) and FTIR spectroscopy.

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UNIVERSITY OF PISA

Department of Chemistry and Industrial Chemistry

In collaboration with

Master Degree in Chemistry Curriculum: Analytical Chemistry

“Characterization of nanomaterials, bovine serum albumin and

their mixtures using a dynamic surface tension detector (DSTD),

dynamic light scattering (DLS) and FTIR spectroscopy”

Candidate: Manuela Marongiu

Internal Supervisor: Prof.ssa Stefania Giannarelli External Supervisor: Dr.ssa Emilia Bramanti Examiner: Dr. Luca Bernazzani

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“A scientist in his laboratory is not a mere technician:

he is also a child confronting natural phenomena that

impress him as though they were fairy tales”

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Abbreviations

AES Auger Electron Spectroscopy

AFM Atomic Force Microscopy

Ag-NPs Silver Nanoparticles

ATR Attenuated Total Reflectance

BSA Bovine Serum Albumin

CD Circular Dichroism

CE Capillary Electrophoresis

CEA Alternative Energies and Atomic Energy Commission

CNTs Carbon NanoTubes

CV% Coefficient of Variation percentage

DLS Dynamic Light Scattering

DSTD Dynamic Surface Tension Detector

EHS Environmental Health and Safety

FIA Flow Injection Analysis

FSD Fourier Self Deconvolution

FTIR Fourier Transform Infrared Spectroscopy

HPLC High Performance Liquid Chromatography

HSA Human Serum Albumin

ITC Isothermal Titration Calorimetry

JRC Joint Research Center

LC-MS Liquid Chromatography Mass Spectroscopy

MAS-NMR Magic Angle Spinning Nuclear Magnetic Resonance

MW Molecular Weight

NMR Nuclear Magnetic Resonance

NMs Nanomaterials

NPs Nanoparticles

PBS Phosphate Buffered Saline

PEEK PolyEther Ether Ketone

pI Isoelectric point

SEC Size Exclusion Chromatography

SEM Scanning Electron Microscopy

SERS Surface Enhanced Raman Spectroscopy

SPR Surface Plasmon Resonance

TEM Transmission Electron Microscopy

TGS TriGlycine Sulfate

TiO2-NPs Titanium Dioxide Nanoparticles

TOF-MS Time-Of-Flight Mass Spectroscopy

UV-Vis Ultraviolet–Visible spectroscopy

UW Ultrapure Water

XANES X-ray Near Edge Absorption Spectroscopy

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Abstract

The widespread use of nanomaterials in consumer products increases the probability of exposure to humans and the environment.

Interaction of nanoparticles with proteins is the basis of nanoparticle bio-reactivity. This interaction gives rise to the formation of a dynamic nanoparticle-protein corona. The protein corona may influence cellular uptake, inflammation, accumulation, degradation and clearance of the nanoparticles. Furthermore, the nanoparticle surface can induce conformational changes in adsorbed protein molecules, which may affect the overall bio-reactivity of the nanoparticle.

This work has been developed in the framework of NANoREG project, which is the first FP7 project to get information on Environmental Health and Safety (EHS) issues of nanomaterials by the scientific evaluation of available data and new test methods.

In order to understand the biological safety of nanoparticles, the interactions of silver nanoparticles (Ag-NPs) and titanium dioxide nanoparticles (TiO2-NPs) with bovine serum albumin (BSA) were

investigated by applying a multi-technique approach.

In this thesis, a multidimensional Dynamic Surface Tension Detector (DSTD) is presented in a novel Flow Injection Analysis (FIA) application to the characterization of Ag-NPs, TiO2-NPs, BSA

and their mixtures. Dynamic Light Scattering (DLS) and Fourier Transform Infrared Spectroscopy (FTIR) were also used to implement DSTD data.

DSTD has been successfully applied to the study of surface activity of nanofluids and nanofluids/protein mixtures. FTIR spectroscopy has been used to identify the structural changes of proteins induced by the interaction with NPs, and DLS has been used to determine the size of particles.

DSTD and DLS measurements confirmed that the nanoparticles aqueous solutions tend to aggregate, unless surfactants or BSA itself are present as dispersant, in agreement with the literature. The tendency to form aggregated species was also observed for BSA in water. The data obtained by FTIR spectroscopy clearly showed that BSA structure in water is not native.

Both DSTD, DLS and FTIR data showed that BSA and NPs interact. FTIR showed that BSA interacting with nanoparticles assumed random or ordered structures (-helix and -sheets) depending on the size of NPs aggregates and NPs/BSA ratio. When big NPs aggregates are present in solution, as described by DLS data, it seems that adsorbed BSA is unfolded assuming a random coil. When smaller NPs are present the adsorbed BSA has an ordered α/β conformation. Both random structures and β-sheets give an increase of surface activity, except for protein aggregates, in agreement with DSTD data.

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Chapter 1 Introduction and aim of the thesis

Nanoscience is a new branch that spans fields of physics, chemistry and materials science. Nanoparticles are widely employed in electronics, catalysis, sensors and coatings but their interest reaches also the environmental sciences, biotechnology and medicine. The special properties of nanomaterials arise from their size, which is between 1 and 100 nm by definition. All material properties (e.g. mechanical, electrical, optical, thermal, etc.) ultimately result from the constituting atoms as well as their bonds and their arrangement. Then, their characterization and the understanding of their properties is very important. When dissolved in a solvent, nanoparticles give nanofluids. Nanofluids are liquids containing nanoparticle, nanotube or nanowire suspensions. Due to their unique properties, nanofluids are relevant for an array of applications including micro-channel cooling of integrated circuits, manufacturing, biomedical therapies and diagnosis, and pharmaceutical and chemical processing. They have been shown to exhibit novel properties as compared with pure liquids including increased thermal conductivity, high single-phase and boiling heat transfer coefficients, and increased critical heat flux [1-8].

The vapor-liquid interfacial tension, in particular, plays a major role in applications related to boiling and condensation. However, it is not clear how surface tension may change with particle parameters such as size, and concentration in nanofluids. Significant efforts have been devoted towards defining and measuring the contact angle between a liquid and micro- and nanoparticles [9-13] and on establishing a theoretical framework to link the properties to the modeling equations governing the contact angle. However, the capillary properties of nanofluids such as the contact angle of sessile droplets and the effective vapor–liquid interfacial tension of the nanofluid have been rarely investigated [14-16].

The effect of NPs on the human body is of increasing concern as these particles are found in an ever-expanding variety of goods. Humans are exposed to NPs through inhalation, ingestion, dermal contact and injection. The small size of NPs facilitates their uptake into cells, as well as transcytosis across epithelial cells into blood and lymph circulation to reach sensitive target sites where they then persist. Although many studies have been done on TiO2 toxicity in animal models

and cell cultures [17-20], the study of the potential risks presented by NPs is still unknown and immediately needed.

Another important aspect of the interaction between nanoparticles/nanotubes and biological systems is the evaluation of the capability of nanostructured surfaces to interfere with the aggregation process of proteins. Protein and peptide aggregation into characteristic oligomer and amyloid fibrils has been, indeed, recognized as the major cause of various neurodegenerative diseases, such as Alzheimer's, Parkinson's, diabetes type 2, etc. [21]. The results obtained from

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many investigations showed that nanoparticles interfere with aggregation process of proteins. However, in several cases promote the amyloid fibrils formation, in other cases retard or inhibit completely this process [22-28].

This work has been developed in the framework of NANoREG project, which is the first FP7 project aimed to get information on Environmental Health and Safety (EHS) issues of nanomaterials by the scientific evaluation of available data and new test methods.

The aim of this thesis is the characterization of nanomaterials using a Dynamic Surface Tension Detector (DSTD) and their interactions with Bovine Serum Albumin (BSA). The nanomaterials investigated were silver nanoparticles (Ag-NPs) and titanium dioxide nanoparticles (TiO2-NPs).

Two other techniques were used to implement DSTD data: Dynamic Light Scattering (DLS) and Fourier Transform Infrared Spectroscopy (FTIR). The BSA is a well characterized, good model protein. In solution the BSA molecule presents a versatile conformation modified by change in pH, temperature, ionic strength, presence of ions and ligands, which influence the protein structure and properties [29]. More importantly, BSA is an adequate model system for the study of nanoparticle/protein interactions because in mammals, serum albumin is one of the most abundant plasma proteins and has a role in maintaining colloid osmotic pressure (needed for proper distribution of body fluids between intravascular compartments and body tissues), binding (in which it acts as a plasma carrier by non-specifically binding several hydrophobic steroid hormones) and transport (of hem in and fatty acids) [30].

The DSTD is a drop-based analyzer that, when used as a chromatographic detector, provides real-time dynamic surface pressure measurements of components as they elute. The DSTD has evolved from an optical measurement-based instrument to a pressure sensor-based instrument, and has been used in conjunction with Flow Injection Analysis (FIA) and High Performance Liquid Chromatography (HPLC) [31-38]. More recently, a novel DSTD calibration procedure has been presented and applied, based on a dual-mobile phase calibration procedure that allows the analyst to apply different mobile phases for the analyte (e.g. denatured protein) and the calibration standard [39-41].

DSTD has been widely applied to the study of surface activity of proteins [39-45]. Protein adsorption at the vapor–liquid interfaces is important, indeed, in a number of processes. However, it has never been applied to the characterization of nanofluids, nor to the study of nanofluids/protein mixtures.

DLS is an important experimental technique in science and industry. This technique is one of the most popular methods used to determine the size of particles. It is applicable in the range from

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about 0.001 to several microns, which is difficult to achieve with other techniques. The knowledge on size, shape and morphology of the particles is of great importance for the study of protein-nanoparticle interactions. These parameters, namely, influence the properties of proteins and nanoparticles, their solubility, stability, distribution in the solution and eventually in the biological matrices and, indirectly, also the technology of their production [46].

Infrared spectroscopy is one of the classical methods for the structure determination of small molecules. In particular, in protein chemistry, FTIR, due to its sensitivity to the chemical composition and architecture of molecules, is a valuable tool for the investigation of protein conformation and protein folding, unfolding and misfolding.

As the interaction between nanoparticles and proteins may alter protein function, the study of this topic is important for safety issues as well as for biotechnological and medical applications.

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Chapter 2 State of the art

2.1 Nanomaterials.

Nanomaterials (NMs) and nanotechnology are a prosperous industrial sector that for since some years, feeds a market becoming bigger [47]. NMs are not only employed in modern and cutting-edge technology but also in the common products widely used. In particular, the nanoparticles of titanium dioxide (TiO2-NPs), whose annual production is estimated to be around 5000 tones [48],

represent 70% for the realization of cosmetics and sunscreens [49]. The Ag-NPs, although not in an amount equaling the annual production of TiO2-NPs, are still present in a number of different

products, especially in the food area. In addition, many other metal nanomaterials or their oxides are now commonly used in industrial sectors such as: cosmetics, construction, energy, textile, medical, pharmaceutical and food. The metal nanoparticles are derived from the mining activity, they include metals and semimetals. May be constituted by individual elements (Ag, Si, Au, Cu, Pt, Fe, Al, Ni, B, Co, etc.) or from their compounds (SiC, Si3N4, WC, etc.). Without doubt the most

important sub-category of this class includes metal oxides (Al2O3, TiO2, ZnO, CeO2, CuO, SnO2,

Fe2O3, MnO, ZrO2, etc.) which, to date, are produced in large quantities because it fit into many

everyday products. As a matter of fact their list is very long and their classification criterion very articulate, enabling to distinguish them on the basis of the different methods of preparation, the size, degree of purity, etc [50]. Furthermore, in addition to inorganic NM, organic NM are widely employed, such as fullerenes, Carbon NanoTubes (CNT) and Carbon Black (CB). These data suggest that these NM can cause a serious environmental impact generated by their inevitable dispersion in the various compartments, and are also sufficient to raise doubts regarding the effects of NM on human health and other living being [51, 52].

In the last five years, there has been a continuous increase in the production of ecotoxicological studies to understand the effects of NM. However, the increasing demand of NM for the market makes their use massive and faster than the development of effective, reproducible and standardized toxicity assays for the assessment of NM effects.

This gap has important regulatory issues. Currently, there is a delay in the production of specific test results such as concentration-response curves, EC50 values, threshold values, etc. Moreover, manufacturers are not required to declare sources of NM in their products.

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2.1.1 Silver nanoparticles (Ag-NPs).

Ag-NPs (Figure 2.1) are the most important NPs because of their applications. These nanoparticles have many important applications that include spectrally selective coating for solar energy absorption and intercalation material for electrical batteries, as optical receptors, polarizing filters, catalysts in chemical reaction, biolabelling and as antimicrobial agents. Ag-NPs are also used for their optical properties in diagnosis and imaging [53, 54]. Som et al. state that the Ag-NPs are also used to cover the facades of buildings. It is especially the textile sector, however, to absorb most of the production of Ag-NPs with percentages ranging from 12% to 50% [55]. Currently, in China new experiments are in progress to treat rice crop with Ag-NPs as a pesticide and antiparasitic [56].

Figure 2.1. TEM characterization of Ag-NPs material [57].

Application of silver nanoparticles in these fields is dependent on the ability to synthesize particles with different chemical composition, shape, size, and monodispersity. There are several procedures for the synthesis of silver nanoparticles in aqueous suspension, based essentially on the reduction of Ag+ to elemental silver. It is worth remembering: i) the reduction by citric acid, sodium borohydride; ii) the reduction by radiation; iii) the reduction by ultrasound; iv) the reduction by biological methods (nitrate reductase); v) the reduction by electrochemical methods (electrolysis of solution). In some chemical methods, a stabilizer (surfactant) is added to the first solution to prevent agglomeration of Ag-NPs, whereas in biological methods there is no need to add a stabilizing agent.

Together with their useful applications in many area, there is increasing concerning related to the biological impacts of the use of silver nanoparticles on a large scale, and the possible risks to the environment and health. In this scenario, some recent studies have been published based on the investigation of potential inflammatory effects and diverse cellular impacts of silver nanoparticles. Another important issue related to nanoparticle toxicity in biological media is their capacity to damage the genetic material, since nanoparticles are able to cross cell membranes and reach the cellular nucleus. In this regard, there is increasing interest in the analysis of potential nanoparticle genotoxicity, including the effects of different nanoparticle sizes and methods of synthesis. However, little is known about the genotoxicity of different silver nanoparticles and their effects on the DNA of organisms; thus further studies in this field are required [58].

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2.1.2 Titanium dioxide nanoparticles (TiO

2

-NPs).

TiO2 (Figure 2.2A) is a white pigment and because of its brightness and very high refractive index

it is most widely used [59]. Titanium dioxide occurs in nature as the well-known minerals rutile (an tetragonal mineral), anatase (an tetragonal mineral) and brookite (an orthorhombic mineral). Rutile and anatase are shown in Figure 2.2B. Approximately four million tons of this pigment are consumed annually worldwide. In addition, TiO2 accounts for 70% of the total production volume

of pigments worldwide, and is in the top five NPs used in consumer products. TiO2 can be used in

paints, coatings, plastics, papers, inks, medicines, pharmaceuticals, food products, cosmetics, and toothpaste. It can even be used as a pigment to whiten skim milk. TiO2-NPs are also used in

sunscreens. In addition, TiO2 has long been used as a component for articulating prosthetic

implants, especially for the hip and knee. These implants occasionally fail due to degradation of the materials in the implant or a chronic inflammatory response to the implant material. Currently, TiO2-NPs are produced abundantly and used widely because of their high stability, anticorrosive

and photocatalytic properties. Some have attributed this increased catalytic activity to TiO2-NPs to

their high surface area, while others attribute it to TiO2-NPs being predominantly anatase rather

than rutile. TiO2-NPs can be used in catalytic reactions, such as semiconductor photocatalysis, in

the treatment of water contaminated with hazardous industrial by-products, and in nanocrystalline solar cells as a photoactive material. Industrial utilization of the photocatalytic effect of TiO2-NPs

has also found its way into other applications, especially for self-cleaning and anti-fogging purposes such as self-cleaning tiles, self-cleaning glass, self-cleaning textiles, and anti-fogging car mirrors. In the field of nanomedicine, TiO2-NPs are under investigation as useful tools in advanced

imaging and nanotherapeutics. For example, TiO2-NPs are being evaluated as potential

photosensitizers for use in PhotoDynamic Therapy (PDT). In addition, unique physical properties make TiO2-NPs ideal for use in various skin care products. Nano-preparations with TiO2-NPs are

currently under investigation as novel treatments for acne vulgaris, recurrent condyloma accuminata, atopic dermatitis, hyperpigmented skin lesions, and other nondermatologic diseases. TiO2-NPs also show antibacterial properties under UV light irradiation [60].

(A) (B)

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2.2 Bovine serum albumin (BSA).

Serum albumin is a globular protein (Figure 2.3). It is the most abundant protein in the circulatory system. BSA typical blood concentrations is 5 g/100 mL, 80% contributing to colloid osmotic blood pressure. Bovine Serum Albumin (BSA) molecular mass is 66 kDa and its isoelectric point is 4.7 [45]. The primary structure is built from 583 amino acid residues containing 20 Tyr and 35 Cys residues, 34 of which are involved in 17 S–S bridge, it is characterized by an overall oblate shape, and it consists of three domains (I, II, and III), each stabilized by an internal network of disulphide bonds and each bearing a number of ionizable groups with opposite signs.

Figure 2.3. Bovine Serum Albumin (BSA).

In addition, it has now been determined that albumin is chiefly responsible for the maintenance of blood pH and it is located in every tissue and bodily secretion, with the extracellular protein comprising 60% of total albumin. The most important property of albumin is its ability to bind reversibly an incredible variety of ligands and it is the principal carrier of fatty acids that are otherwise insoluble in circulating plasma. But albumin performs many other functions as well, such as sequestering oxygen free radicals and inactivating various toxic lipophilic metabolites. Although albumin has a broad affinity for small negatively charged aromatic compounds, it has high affinities for fatty acids, hematin, and bilirubin. Additionally, it forms covalent adducts with pyridoxyl phosphate, cysteine, glutathione, and various metals, such as Cu(II), Ni(II), Hg(II), Ag(II), and Au(I). The participation of albumin as the key carrier or reservoir of nitric oxide [62], which is implicated in a number of important physiological processes, including neurotransmission, serves to illustrate further the continuing recognition of the utility of albumin. To date, albumin is the most multifunctional transport protein known [63]. The substantial information on serum albumin has led to some contradictory results and discussions. Based largely on hydrodynamic experiments [64-66] and low-angle X-ray scattering [67], serum albumin was postulated to be an oblate ellipsoid with dimensions of 140x40x40 Å (where a = b < c). Experiments have continued to support these dimensions [68, 69] and using a different variety of data, it has been developed a model of albumin having the shape of a cigar [70].

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However, 1H NMR studies indicated that an oblate ellipsoid structure of albumin was unlike and a heart-shaped structure was proposed (Figure 2.3) [71]. This was in agreement with X-ray crystallographic data [72]. Previous studies, performed with FTIR and circular dichroism, indicated that the secondary structure contained about 68% - 50% α-helix and 16% -22% β-sheet [73-78]. Modifications in the secondary as well as tertiary structures of BSA occur in dependence of pH, temperature, and various kinds of denaturants [42, 73]. Foster [76] reported BSA has several isomeric forms at different pH media that correspond to different α-helix contents. Conformers are classified as E (extended at pH 2.7, 35% α-helix), F (fast migration at pH 4.5, 45% α-helix), N (normal dominant form at neutral pH, 55% α-helix), B (basic form at pH 10, 48% α-helix) and A (aged at pH 10, 48% α-helix). Riley and Arndt [79, 80] suggested that thermally denatured bovine serum albumin has probably the same fundamental type of folding of the polypeptide chains as the native one, which is 55% α-helix and 45% random conformation from X-ray scattering studies. Harmsen and Braam [81] concluded that alkali or heat denaturation caused a partial loss of α-helical structure with β-sheet formation. Lin and Koenig [82] investigated heat, acid and alkali denaturation of BSA by Raman spectroscopy and found that heating to 70 °C or a change in pH to below 5 or above 10 caused a decrease in the α-helix content accompanied by an increase in β-sheet.

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2.3 Interaction of nanomaterials and biological systems.

The interactions of nanomaterials with biological systems are inevitable. To elucidate how nanomaterials affect organs and physiological functions, a thorough understanding of how nanomaterials perturb cells and biological molecules is required (Figure 2.4) [83].

Figure 2.4. Interactions of nanoparticles with biological systems at different levels. Nanoparticles enter the human body through various pathways, reaching different organs and contacting tissues and cells. All of these interactions are based on nanoparticle−biomacromolecule associations.

Rapidly accumulating evidence indicates that nanomaterials interact with the basic components of biological systems, such as proteins, DNA molecules, and cells [84-86]. The driving forces for such interactions are quite complex and include the size, shape, and surface properties of nanomaterials [87-90]. These observations not only support the hypothesis that basic nano−bio interactions are mainly physicochemical in nature but also provide a powerful approach to control the nature and strength of a nanoparticles interactions with biological systems. The literature regarding nanoparticle−biological system interactions has increased exponentially in the past decade (Figure 2.5).

Figure 2.5. Analysis of literature statistics indicates growing concern for nanoparticles. The number of publications and citations was obtained using the keywords “nanoparticles” and “biological systems” in the subject area of “Chemistry” when searching the Thomson Reuters (ISI) Web of Knowledge for the period 1995−2013 [83].

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However, a mechanistic understanding of the chemical basis for such complex interactions is still lacking. Accumulating experimental evidence suggests that nanoparticles interact with biological systems at nearly every level, often causing unwanted physiological consequences [83]. The real identity of nanoparticles in biological systems is determined by their intrinsic properties, such as their size, shape, surface charge, and hydrophobicity (Figure 2.6).

Figure 2.6. Factors influencing nanoparticle−biomolecule interactions.

Therefore, a rigorous characterization of nanoparticles is essential prior to investigate nano-bio interactions [83].

Nanoparticles enter the human body via multiple routes, encountering various biomolecules regardless of the pathway by which they enter the human body. The interactions of nanoparticles with biological system occur at various levels:

 nanoparticle-phospholipid interactions, especially in pulmonary surfactant solutions;  interactions between nanoparticles and membranes;

 nanoparticle-protein interactions;

 interactions between nanoparticles and proteomes;  nanoparticle-DNA interactions;

 adsorption of crucial small molecules by nanoparticles;

 nanoparticle-cell interactions (cellular uptake and intracellular transport, cellular sub-structural and functional alterations, cell membrane disruption, ion channel inhibition, cytoskeleton alterations, mitochondrial and nuclear alterations);

 alteration of the oxidative stress (induced or reduced, systematic cellular biochemical perturbations, perturbation of cell signaling pathways, global biochemical alterations).

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In this work, we would focus on nanoparticle-protein interaction.

The interactions between nanoparticles and proteins are complex. Nanoparticles are, generally, surrounded by plasma proteins upon entering the circulatory system. Upon contact with body fluids, nanomaterials are immediately covered with proteins [91-93]. Rapidly forming protein corona determines the nanoparticles physicochemical properties, including hydrodynamic size, surface charge, and aggregation behavior [94-96]. Furthermore, the interaction with cell membranes and the mechanism of cellular uptake is meticulously controlled by the adsorbed proteins. Therefore, the corona defines the biological identity of nanoparticles, influencing relevant parameters including cytotoxicity [97, 98], body distribution, and endocytosis into specific cells [99-102].

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2.4 Protein Corona.

2.4.1 Structure and composition of “corona”.

The structure and composition of the protein corona depends on the physic-chemical properties of the nanomaterials (size, shape, curvature, composition, surface functional groups, hydrophobicity and surface charges), the nature of the physiological environment (blood, interstitial fluid, cell cytoplasm, etc.), and the duration of exposure [103]. The protein corona alters the size and interfacial composition of a nanomaterial, giving it a new biological identity. The biological identity determines the physiological response including agglomeration, cellular uptake, circulation lifetime, signaling, kinetics, transport, accumulation, and toxicity. Protein corona is complex and there is no one “universal” plasma protein corona for all nanomaterials. Thus, the composition of the protein corona is unique to each nanomaterial and depends on many parameters [103].

The majority of adsorbed biomolecules on the surface of nanoparticles in blood plasma are proteins, and recently some minor traces of lipids have also been reported. The adsorption of proteins on the surface of nanoparticle is governed by protein–nanoparticle binding affinities as well as protein–protein interactions. Proteins that adsorb with high affinity form what is known as the “hard” corona, consisting of tightly bound proteins that do not readily desorb, and proteins that adsorb with low affinity form, the “soft” corona, consisting of loosely bound proteins (Figure 2.7).

Soft and hard corona can also be defined based on their exchange times. Hard corona usually

shows much larger exchange times in the order of several hours [104]. A hypothesis is that the hard corona proteins interact directly with the nanomaterial surface, while the soft corona proteins interact with the hard corona via weak protein–protein interactions [105].

Figure 2.7. Schematic illustration of soft and hard protein corona and the concept of the rate of adsorption and desorption which determines the exchange time and lifetime of proteins in the protein corona. The hard or soft corona is not composed of only a single protein; in this scheme, the complexity of the presence of different proteins is not shown.

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The thickness of protein corona can due to many parameters such as protein concentration, particle size, and surface properties of particle. Most plasma proteins present a hydrodynamic diameter of about 3–15 nm.

A model for the protein corona has been proposed by Simberg et al. [106]; it consists of “primary binders” that recognize the nanomaterial surface directly and “secondary binders” that associate with the primary binders via protein–protein interactions. Such a multilayered structure is significant for the physiological response as the secondary binders may alter the activity of the primary binders or “mask” them, preventing their interaction with the surrounding environment. The competitive adsorption of proteins on the limited surface of nanoparticles containing the collective effects of incubation time, concentration of protein, and adsorption affinity between protein and nanoparticle surface is called “Vroman effect” [107, 108].

Hard Corona. One of the mechanisms of adsorption of proteins on the surface of nanoparticles is

the entropy-driven binding. The adsorption mechanism for entropy-driven-bonded proteins such as fibrinogen, lysozyme, ovalbumin, and human carbonic anhydrase II is the release of bound water from the surface of the nanoparticle. In this case, the increase in entropy due to the released water molecules is larger than the decrease in the entropy due to the adsorption of proteins [109].

Soft Corona. The molecules which are loosely bonded to the nanoparticle surface or have weak

interaction with the hard corona form the soft corona. In the case of some nanoparticles, especially those with a preformed functional group such as pegylated nanoparticles, there is only a weak corona covering the surface and no hard corona is observed. The theoretical challenge of understanding why certain proteins are adsorbed in a competitive manner is unsolved [110].

2.4.2 Protein conformation.

During adsorption on the nanoparticles, proteins may undergo structural rearrangements called “conformational changes” [103]. These changes are thermodynamically favorable if they allow a hydrophobic or charged sequence within a protein to interact with a hydrophobic or charged nanomaterial surface, respectively. Conformation of adsorbed proteins is altered more in the presence of charged or hydrophobic nanomaterials. Binding of proteins to planar surfaces often induces significant changes in secondary structure, but the high curvature of NPs can help proteins to retain their original structure. However, study of a variety of NP surfaces and proteins indicates that the perturbation of protein structure can appear. Lysozyme adsorbed onto silica NPs or bovine serum albumin adsorbed on Au-NPs surfaces showed a rapid conformational change at both secondary and tertiary structure levels. Most of the studies have reported that loss of α-helical content occurs as detected by circular dichroism spectroscopy when proteins are adsorbed onto NPs and a significant increase in sheet and turn structures [103].

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In some cases, the study of the interactions was focused on the effect of the curvature of nanostructured surfaces on the secondary structure of proteins adsorbed on nanoparticles/nanotubes using CD, FTIR and fluorescence spectroscopy [111-117].

Another important aspect of the interaction study between protein and nanoparticles/nanotubes is the evaluation of the capability of nanostructured surfaces to interfere with the aggregation process of protein adsorbed because protein and peptide aggregation into characteristic amyloid fibrils is a major cause of various neurodegenerative diseases such as Alzheimer's, Parkinson's, diabetes type 2, etc. [21]. The results obtained from many investigations showed that nanoparticles interfere with aggregation process of proteins but in some cases promote the amyloid fibrils formation and in other cases retard or inhibit completely this process [22-28].

2.4.3 Dynamic of protein corona and its time evolution.

The competition between more than 3,700 proteins in the blood plasma for adsorption on the surface of the nanoparticle changes the composition of the corona over time [109]. Therefore, corona is not a fix layer, and its composition is determined by the kinetic rate of adsorption and desorption of each protein and lipid (Figure 2.7). In most of the cases, proteins with high abundance in the plasma are adsorbed on the surface, and over the time, they are replaced by proteins with lower concentration but higher affinity. Recently the protein corona formation has been studied on FePt and CdSe/ZnS [118] and Au nanoparticles [119]. The protein absorption has been measured after 5–30 min incubation time, showing that the adsorption of blood serum proteins on an inorganic surface is time dependent. The highest mobility proteins arrive first and are later replaced by less mobile proteins that have a higher affinity for the surface. This process may take several hours. Proteins adsorbed to a nanomaterial are in a continuous state of dynamic exchange. At any time, a protein may desorb, allowing other proteins to interact with the nanoparticle surface. These changes in the composition of the protein corona resulting from desorption/adsorption are known as the “Vroman effect”, as reported above. This effect takes into account that the identities of the adsorbed proteins can change over time even if the total amount of adsorbed protein remains roughly constant [103].

2.4.4 Parameters affecting protein corona.

Various parameters such as nanoparticle size, shape, curvature, surface charge (zeta potential), solubility, surface modification, and route of administration of nanoparticles to the body affect the composition, thickness, and conformation of protein corona [103]. These parameters have been reviewed recently by various groups [96, 109, 110]. Among the nanoparticle parameters which affect the protein corona, the surface properties such as hydrophobicity and surface charge play a

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more significant role than other parameters [108]. In the following section, the role of each parameter is explained with more details. Better understanding of role of each physicochemical parameter on the protein corona is promising for design of targeting nanomaterial, long-circulating drug carriers, or for decreasing the toxicity.

Surface charge of nanoparticle. Nanoparticle surface charge is an important factor in protein

interaction. It has been reported that by increasing the surface charge of nanoparticles, the protein adsorption increases. Positively charged nanoparticles prefer to adsorb proteins with isoelectric points pI < 5.5 such as albumin, while the negative surface charge enhances the adsorption of proteins with pI > 5.5 such as IgG [108]. Surface charge can also denature the adsorbed proteins. In a recent study on the gold nanoparticles with positive, negative, and neutral ligands, it was found that proteins denature in the presence of charged ligands, either positive or negative, but the neutral ligands keep the natural structure of proteins [109].

Nanoparticle material. The study of the plasma proteins bound to Single-Walled Carbon Nano

Tubes (SWCNT) and nano-sized silica indicated different patterns of adsorption. Serum albumin was found to be the most abundant protein coated on SWCNT but not on silica nanoparticle. TiO2,

SiO2, and ZnO-NPs of similar surface charge bind to different plasma proteins (Table 2.1) [120].

Table 2.1. Identification of proteins bound to nanoparticles by gel electrophoresis and mass spectrometry[120].

Nanoparticles Proteins

TiO2

Albumin, fibrinogen (α and β chains), histidine-rich glycoprotein, kininogen-1, complement C9 and C1q, Ig heavy chain (γ), fetuin A, vitronectin, apolipoprotein A1 SiO2

Albumin, fibrinogen (α and β chains), complement C8, Ig heavy chain (γ, κ), apolipoprotein A

ZnO Albumin, Ig heavy chain (α, μ, γ), apolipoprotein A, immunoglobulin (J chain), α-2-macroglobulin, transferring, α-1-antichymotrypsin

Surface functionalization and coatings. Pre-coating and surface functionalization can be

employed to decrease the adsorption of proteins or engineer the protein corona composition. Numerous studies established that aqueous suspensions of non-functionalized nanoparticles are stabilized against agglomeration by the addition of bovine/human serum albumin (BSA/HSA) and some other proteins [103]. The effect has also been exploited in production for the debundling and dispersion of graphene and CNT material. Especially albumins in water or Dulbecco’s Modified Eagle Medium (DMEM) have been used to disperse and stabilize a wide variety of nanomaterials: CNTs, metal nanoparticles, metal carbide nanoparticles, and metal oxide nanoparticles [103].

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Hydrophilicity/Hydrophobicity. The hydrophobicity affects both the amount of adsorbed protein

as well as the composition of protein corona. The enhanced adsorption of proteins on hydrophobic surface in comparison with hydrophilic surface increases the rate of opsonization1 of hydrophobic nanoparticles [108]. Hydrophobic or charged surfaces tend to adsorb more proteins and denature them with a greater extent than neutral and hydrophilic surfaces. Hydrophobic nanoparticles adsorb more albumin molecules than hydrophilic nanoparticles, even though the affinity of the protein to both nanoparticle types is roughly the same [121]. This suggests that hydrophobic copolymer nanoparticles have more protein-binding sites. This may result from “clustering” of the hydrophobic polymer chains, forming distinct “islands” which act as protein binding sites. Therefore, it can be concluded that the affinity of proteins to nanomaterials with uniform surface chemistry tends to increase with increasing charge density and hydrophobicity [122].

Nanoparticle size. Protein-binding affinities for NPs depends on their surface curvature. In

addition to protein-binding affinity, the composition of protein corona is different for same NPs but with different sizes [104]. The change of composition and organization of proteins in the corona is very significant when the nanoparticle size is approaching the size of proteins [109]. The highly curved surfaces of nanomaterials decrease protein–protein interactions. Proteins adsorbed to highly curved nanoparticles tend to undergo fewer changes in conformation than those adsorbed to less curved surfaces. Size and curvature of nanoparticles also appear to affect protein binding. Dobrovolskaia et al. [119] reported that more proteins were adsorbed on 30 nm than on 50 nm gold particles. Another study involving the interaction of gold nanoparticles with common plasma proteins suggests that the thickness of the adsorbed protein layer increases progressively with nanoparticle size. Gold nanoparticles can initiate protein aggregation at physiological pH, resulting in the formation of extended, amorphous protein–nanoparticle assemblies, accompanied by large protein aggregates without embedded nanoparticles. Proteins on the Au nanoparticle surface are observed to be partially unfolded; these nanoparticle-induced misfolded proteins likely catalyze the observed aggregate formation and growth [103].

1

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Table 2.2 summarizes various parameters that can affect the composition, thickness, and conformation of protein layers [105].

Table 2.2. The role of nanoparticles physicochemical and environmental parameters on the protein corona [105].

The parameter The observed effect

Higher charge density of NP

-Increases density and thickness of corona -Charge particle has higher opsonization rate - Increases protein conformal change

Higher hydrophobicity of NP

- Increases the thickness of protein corona - Increases protein conformal change

- Hidrophobicity increases the opsonization rate

Higher curvature of NP

- Increases the corona thickness - Decreases the conformational change

- Does not change the identity of adsorbed proteins Higher protein concentration in environment - Higher thickness

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2.5 Techniques used to study the interaction between BSA

and nanomaterials.

2.5.1 Characterization of nanoparticles.

A very large collection of methods has been used to characterize nanoparticle physical and chemical properties. Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), and Atomic Force Microscopy (AFM) provide information on nanoparticle morphology [123]. Dynamic Light Scattering (DLS) provides information on the hydrodynamic radii of nanoparticles in solution [124]. The surface charge properties of nanoparticles can be determined with δ-potential measurements [125]. Auger Electron Spectroscopy (AES), X-ray Photoelectron Spectroscopy (XPS), Time-Of-Flight Mass Spectrometry (TOF-MS), and elemental analyses reveal chemical composition details of nanoparticles [126, 127]. Surface ligands or adsorbed molecules can be identified with Magic Angle Spinning Nuclear Magnetic Resonance (MAS-NMR), Liquid Chromatography Mass Spectroscopy (LC-MS), and Fourier-Transform Infrared Spectroscopy (FTIR) [128-130]. Surface-Enhanced Raman Spectroscopy (SERS) is also among the most frequently applied analytical method for characterizing nanoparticles [131].

2.5.2 Characterization of protein corona.

Relevant analytical techniques are necessary to study dynamic processes for NP–protein interactions and to characterize the properties of the NP-protein complex, which are crucial to understanding the potential effects of NPs on cells and mechanisms. One hot topic about protein corona is about the formation and evolution process of protein corona and the major composition of adsorbed proteins. A series of analysis methods and techniques such as optical absorption spectroscopy, TEM and DLS, have been used to characterize the protein composition and structure. TEM and DLS are widely used to measure the thickness of the protein corona on NPs in dried samples and in an aqueous solution, respectively [132]. Moreover, UV–vis spectra can detect significant shifts in the peak position of the Surface Plasmon Resonance (SPR) of NPs before and after protein adsorption. The protein adsorption probably broadens the absorption spectra and decreases absorption intensity, which is a rapid and simple method to characterize the dynamic process of NPs/protein interaction [133]. Based on the signals of left and right circularly polarized light, CD spectra are suitable to determine secondary structure of adsorbed proteins on NPs in aqueous solution [134]. When BSA in PBS was mixed with Au-NPs, in the formed BSA/Au-NPs conjugates, the percentage of helical structure significantly decreased. The structure of the α-helix has a characteristic CD signal in the far UV region. After conjugation, a significant decrease

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of ellipticity at the band around 208 nm and 220 nm was observed, which meant that the α-helical structure was destroyed and protein was unfolded after adsorption; when proteins are bound to NPs in biological fluids, the conformation changes with increasing protein concentration [135]. Isothermal Titration Calorimetry (ITC) is also a conventional analytical technique that can explore the thermodynamics parameters, such as binding stoichiometry, binding affinity, and binding enthalpy change [136]. In addition, Nuclear Magnetic Resonance (NMR) has been used to identify and quantify adsorbed proteins and characterize the structure of corona. The interaction changes the resonance angle of incident light and resonance wavelength, which is helpful for studying the dynamic processes of protein adsorption [137]. Conventional analytical techniques like chromatography, Capillary Electrophoresis (CE) [138], are useful for the isolation and identification of proteins in the corona composition. In addition, other analytical methods like Mass Spectrometry (MS) have been used to identify the adsorbed protein profile [139]. To investigate the bound interface structure of proteins, X-ray Near Edge Absorption Spectroscopy (XANES) was used [140]. Novel methods or integrated methodology are urgently required to reveal the interfacial structure of corona and NPs. Promising techniques should not only realize high-throughput screening and identification of proteins in various biological fluids, but also precisely predict potential biological effects quickly, in real time and high resolution [141].

In this work three techniques have been employed in order to study the protein-NPs interaction:  a Dynamic Surface Tension Detector (DSTD) to study the surface tension properties of

NPs and NP-protein mixtures;

 Dynamic Light Scattering (DLS) to get information on the NP/NP-protein size;

 Fourier Transform Infrared Spectroscopy (FTIR) to investigate protein conformational changes.

Very few studies are available in the literature on the surface tension of nanofluids [142-146]. Some recent studies on the surface tension of nanofluids are discussed below. Liu et al. [147] studied stagnation point heat transfer by jet impingement considering the effect of surface tension. Kumar and Milanova [148] measured the surface tension of Single Wall Carbon NanoTube (SWCNT) suspensions in deionized water with the surfactant sodium salt of bis (1-dodecenyl succinamic acid) (NaBDS), using the bubble pressure method. Their results showed that the surface tensions of nanofluids were higher than those of the base fluid.

Zhu et al. [149] measured the surface tension of Al2O3/water nanofluid using the maximum bubble

pressure method. Their results showed that the surface tension of nanofluid was dependent on the temperature and the concentration. They reported an increase of about 5% in the surface tension for a concentration of 1.0 g/L nanofluid over the base fluid. However, they also mentioned that the

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increase was contrary to the findings of other researchers. Radiom et al. [150] measured the surface tension of TiO2/deionized water nanofluid at room temperature using the pendant droplet

method. Their experiments on three different volumetric concentrations of particles showed that surface tension decreased with an increase in particle volume concentration. They also found the surface tension to be a strong function of surfactant concentration. Chen et al. [151] measured the surface tension of three different nanofluids, laponite, Ag and Fe2O3 dispersed in deionized water

[142].

Most authors have determined the surface tension of nanofluids by pendant drop method [143, 151-153]. This method utilizes the Young–Laplace equation to determine the surface tension of the droplet based on the shape of the drop [154]. Bubble pressure method [148, 155] is also used for surface tension determination. Some authors used some other methods for surface tension measurement, Wilhelmy plate method [156, 157] and sessile drop method [158]. Table 2.3 shows a list of experimental processes used [143].

Table 2.3. Different experimental process used in literatures about surface tension of nanofluids [143].

Particle name Diameter [nm] Experimental process

Al2O3 25

Pendant drop method

Laponite 25-30

Silver 10-30

Fe2O3 10-30

CuO 30

Silver < 100

Bubble pressure method

CNTs -

Silica 6-460

Wilhelmy plate method

TiO2 15

Bi2Te3 2.5 and 10.4 Sessile drop method

DSTD is a novel approach in this kind of investigations.

2.5.3 Dynamic surface tension detector (DSTD).

The study and development of the dynamic surface tension detector (DSTD) started upon the pioneering studies of Liggieri and co-workers [159]. DSTD combines the measurement of surface tension with continuous-flow-based liquid sample introduction. Thus, the novelty of DSTD technology is to extend the capillary pressure technique to continuous-flow-based systems like Flow Injection Analysis (FIA) and High Performance Liquid Chromatography (HPLC), thus broadening the scope of capillary drop pressure-based techniques.

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Drops provide an informative and fascinating environment to study chemical and physical phenomena and to develop strategies and instrumentation in chemical analysis [160]. The DSTD has been developed over the past several years by the research groups of Prof. R.E. Synovec (University of Washington, Seattle, WA, USA) and Dr.ssa E. Bramanti (C.N.R., ICCOM-UOS, Pisa, Italy) as a versatile system to detect surface tension in FIA and HPLC experiments, evolving from an optical-measurement-based instrument to a pressure-sensor-based instrument [34, 161, 162]. In the latter configuration, the DSTD is based upon a growing drop method, implementing a pressure sensor where the pressure signal is dependent upon the surface tension properties of a given sample. The DSTD measures the differential pressure as a function of time across the vapor-liquid interface of growing drops that repeatedly form and detach at the end of a capillary tip. The repetitive formation of drops at the end of the capillary sensing tip has significantly enhanced the integration of the DSTD as a detector for FIA and HPLC analytical techniques [33, 163]. The development of a novel calibration technique based on utilizing the ratio of pressure signals acquired from the drop growth of a standard solution and a sample solution (containing the analyte of interest) represented a further improvement of DSTD technology and led to many applications in

the study of the surface activity of polymers and proteins [35, 37]. The DSTD yields information

concerning transfer rates of surface-active molecules from the bulk solution to the vapor–liquid interface and reveals kinetic information with respect to the adsorption processes at the vapor– liquid interface.

The DSTD has been successfully coupled with Size Exclusion Chromatography (SEC) by Miller et al. for the analysis of poly(ethylene glycols) (PEGs) [37]. Also, has been successfully applied to the characterization of proteins both in FIA and in HPLC experiments, which included size exclusion and hydrophobic interaction separation mechanisms [43].

The application of this detector for the characterization of nanoparticles and mixtures BSA/NPs, is completely innovative.

2.5.4 Dynamic light scattering (DLS).

The technique is known by different names. Dynamic light scattering (DLS) is the name that covers different techniques for the measurement of particle size from the dynamic changes of the scattered light intensity. In the DLS technique, the intensity of the scattered light by an ensemble of particles is measured at a given angle as a function of time. The Brownian motion of the dispersed particles determines the rate of change of the scattered light intensity. The temporal intensity changes are converted to a mean translational diffusion coefficient (or a set of diffusion coefficients). Fast intensity changes are related to a rapid decay of the correlation function and a large diffusion coefficient. The diffusion coefficient is then converted into particle size by means of the Stokes-Einstein equation. For this conversion the particles are assumed to be spherical and without

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interaction. Note that colloidal particles in a liquid dispersion contain an attached layer of ions and molecules from the dispersion medium that moves with the particle itself. Therefore, their hydrodynamic particle size is somewhat larger than the size of the particle. DLS has found its place for particle size measurement in the sub-micrometer range, as well for certification of standard reference materials as for industrial and scientific applications. A new application of the technique has become known under the name Diffusing Wave Spectroscopy. Further DLS instrument development may come from the use of new lasers and new detectors and from integration of signals coming from more light beams and detectors. Recently, an instrument has come on the market that applies direct particle tracking. It uses a focused laser to illuminate the particles, which appear as bright moving points under a microscope. The Brownian movement of these points is detected by means of a digital video camera in combination with specific particle tracking software [164].

2.5.5 Fourier transform infrared (FTIR) spectroscopy.

Infrared spectroscopy is one of the classical methods for structure determination of small molecules. Infrared spectroscopy is a valuable tool for the investigation of protein structure, of the molecular mechanism of protein reactions and of protein folding, unfolding and misfolding due its sensitivity to the chemical composition and architecture of molecules. Additional advantages of infrared spectroscopy are its application from small soluble proteins to large membrane proteins, a high time resolution down to 1 μs with moderate effort, often a short measuring time, the low amount of sample required (typically 10-100 μg) and the relatively low costs [165].

The peptide bond between the polypeptide and protein repeat units gives rise to nine characteristic IR absorption bands, namely, amide A, B, and I−VII. Among these, the Amide I and II bands are the two most prominent vibrational bands of the protein backbone. The most sensitive spectral region to the protein secondary structural components is the Amide I band (1700-1600 cm−1), which is due almost entirely to the C=O stretch vibrations of the peptide bonds (approximately 80%).

The frequencies of the Amide I band components are found to be correlated closely to each secondary structural element of the proteins. The Amide II band derives mainly from in-plane N-H bending (40-60% of the potential energy) and from the C-N stretching vibration (18-40%), showing much less protein conformational sensitivity than its Amide I counterpart.

Other amide vibrational bands are very complex depending on the details of the force field, the nature of side chains and hydrogen bonding, which therefore are of little practical use in the protein

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conformational studies. The characteristic IR bands of the proteins and peptides are listed in Table 2.4 [165-167].

Table 2.4. Characteristic infrared bands of peptide bond [149-151].

Designation Approximate frequency (cm-1) Description

Amide A 3300 NH stretching

Amide B 3100 NH stretching

Amide I 1600-1690 C=O stretching

Amide II 1480-1575 CN stretching, NH bending

Amide III 1229-1301 CN stretching, NH bending

Amide IV 625-767 OCN bending

Amide V 640-800 Out-of-plane NH bending

Amide VI 537-606 Out-of-plane C=O bending

Amide VII 200 Skeletal torsion

High sensitivity to small variations in molecular geometry and hydrogen bonding patterns makes the Amide I band uniquely useful for the analysis of protein secondary structural composition and conformational changes. In the Amide I region (1700−1600 cm−1), each type of secondary structure gives rise to a somewhat different C=O stretching frequency due to unique molecular geometry and hydrogen bonding pattern. However, the observed Amide I bands of proteins are usually a convolution of overlapped underlying component bands, which lie in close proximity to one another and are instrumentally unresolvable. Thus, mathematical methods such as resolution-enhancement technique are necessary to resolve the individual band component corresponding to specific secondary structure. Mathematical data analysis methods can be used to “enhance” the resolution of the protein spectrum, allowing the intrinsically broad components to be narrowed and separated beyond the instrument resolution. The mathematical band-narrowing process does not actually increase the instrumental resolution, but rather increases the degree of separation by narrowing the half-bandwidth of individual components for easier visualization. This band-narrowing process is achieved at the expense of spectral quality of the original band, which leads to a degradation of signal-to-noise ratio.

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Several methods have been developed to estimate quantitatively the relative contributions of different types of secondary structures in proteins from their IR Amide I band in solution, including Fourier Self Deconvolution (FSD) curve fitting, second derivative analysis, partial least-squares analysis, and data basis analysis. The FSD-curve fitting and second derivative analysis are the two most popularly used methods [166].

The quantitative analysis of protein secondary structure is based on the assumption that the protein IR absorption can be considered a linear sum of the absorption of few fundamental secondary structural elements. The comparisons of IR spectra with high-resolution X-ray crystal structures of proteins could establish necessary spectra-structure correlations. Over the years, many correlations between IR spectra and particular protein structure have been established and the Amide I band components were assigned by studying the frequency behaviour in proteins and peptides in which the protein secondary structure was known by other techniques [73, 167].

The Amide I band (1700–1600 cm−1) is due mainly to the C=O stretching vibration (approximately 80%) of the amide groups, as stated above, coupled with little in-plane NH bending (< 20%). The exact frequency of this vibration band depends on the nature of hydrogen bonding involving the C=O and N-H moieties [167]. In turn, this is determined by the secondary structure adopted by the polypeptide chain, reflecting the backbone conformation and hydrogen-bonding pattern. Thus, the observed Amide I band contours of proteins or polypeptides consist of overlapping component bands, representing α-helices, β-sheets, turns and random structures.

The assignments of the Amide I band component to each secondary structure element are available for proteins in both H2O and D2O media. The characteristic IR bands of the proteins and peptides

with different secondary structures are listed in Table 2.5 [165].

Table 2.5. Assignment of Amide I band positions to secondary structure [168].

Secondary structure

Band position in H2O/cm -1

Band position in D2O/cm -1

Average Extremes Average Extremes

α-helix 1654 1648-1657 1652 1642-1660 Β-sheet 1633, 1684 1623-1641, 1674-1695 1630, 1679 1615-1638, 1672-1694 Turns 1672 1662-1686 1671 1653-1691 Disordered 1654 1642-1657 1645 1639-1654

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The Table 2.5 is based on the experimental data and assignments of various authors collected and evaluated by Goormaghtigh et al. [169]. In similar tables a discrimination between parallel and antiparallel (ap) β-sheet can sometimes be found, because theory predicts no high wave number component for infinite parallel β-sheets [170].

It is known that in ap-β-sheet structures, the Amide I region displays two typical components, the major one is around 1630 cm-1 and the minor one around 1695 cm-1 . The 1695/1630 ratio has been suggested to be proportional to the percentage of ap-β-strands in a β-sheet. Parallel (p) β-sheets show in the Amide I region only the major component at 1630 cm-1. The broad component at 1646–1648 cm-1 is attributed to random chains, and it is characterized by a wide bandwidth. Indeed, in random structures hydrogen bonds are more exposed, and the presence of water molecules as well as adjacent amino acids gives additional strong interactions. These contribute to the distortion of the structures and consequently to an increase in the bandwidth. The component at 1676–1678 cm-1 is determined by turns connecting β-strands [171].

The sensitivity of the Amide I vibration to secondary structure allows the study of protein folding, unfolding and aggregation. While the folded protein exhibits a structured Amide I spectrum after band narrowing techniques have been applied, the unfolded protein shows a broad, featureless Amide I band centred near 1650 cm−1 which is characteristic of unordered structure. In contrast, aggregated protein often shows a band near or below 1620 cm−1 which is characteristic of intermolecular β-sheets [165]. Infrared spectroscopy was one of the earliest experimental methods used to evaluate the secondary structure of amyloid aggregates responsible for neurodegenerative disorders such as Huntington’s and Alzheimer’s diseases that show a fibrillar morphology. Fibrils showed a predominant parallel β-sheet structure and a small percentage of α-helix [171] and it was shown that some nanoparticles can inhibit amyloid aggregation [172-174].

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Chapter 3 Experimental

3.1 Materials.

BSA (A-8531, 05470) was purchased from Aldrich–Sigma Chemical Co and was used without further purification for all the experiments.

In this work we studied the interaction of BSA with the following nanomaterials:

1) NM-300 nano-Silver < 20 nm reference nanomaterial (Ag-NPs) were provided by the JRC (Joint Research Center; Ispra, Italy) in the framework of NANOGENOTOX joint action. Ag-NPs were used in a variety of studies and projects including the OECD WPMN Sponsorship Programme. The material is a nano-Silver colloidal dispersion with nominal silver content of 10 w/w%. The NM-300 sample dispersion is yellow-brown; it is an aqueous dispersion of nano-Silver with stabilizing agents, consisting of 4% w/w% each of Polyoxyethylene Glycerol Trioleate and Polyoxyethylene (20) Sorbitan mono-Laurat (Tween 20). Ag-NPs were distributed by the Fraunhofer Institute for Molecular Biology and Applied Ecology, Schmallenberg (Germany).

2) Three TiO2 nanomaterials used were provided by the JRC (Joint Research Center; Ispra, Italy) in

the framework of NANOGENOTOX joint action and are named according to their generic codes also used within OECD (Organization for Economic Co-operation and Development) projects (NM100, NM101, NM103). Table 3.1 shows characteristics of nanomaterials used in this work.

Table 3.1. Characteristics of TiO2 nanomaterials: NM100, NM101 and NM103.

Material code Core material Polymorph Particle type Diameter (nm) Inorganic coating Organic coating

NM100 TiO2 anatase nanoparticle 110 Fe,Si,Al no

NM101 TiO2 anatase nanoparticle 6 Si,Al yes

NM103 TiO2 rutile nanoparticle 24.7 Al,Si,Fe yes

The phosphate buffer solutions (PBS) were prepared from monobasic monohydrate sodium phosphate and dibasic anhydrous potassium phosphate (BDH Laboratory Supplies, Poole, England). Ultrapure water (UW) was prepared with an Elga Purelab-UV system (Veolia Environnement, Paris, France).