Detection and characterization of exosomal biomarkers via noble metal nanoparticles conjugation

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Detection and characterization of exosomal biomarkers via

noble metal nanoparticles conjugation

Scipioni Lorenzo

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List of Figures

2.1. Amino acids general structure . . . 13

2.2. Peptide bond formation . . . 14

2.3. Structure of common amino acids . . . 15

2.4. EDC/NHS Reaction . . . 16

2.5. Schematic representation of the 2D distribution of the gold-thiol bond strength . . . 17

2.6. Schematic antibody structure . . . 18

2.7. Schematic representation of plasmon oscillation for a sphere . . . 20

2.8. TEM image of 30 nm gold nanoparticles sample . . . 23

2.9. Schematic structure of pep-K hexapeptide . . . 25

2.10.Schematic representation of hexapeptide coated nanoparticles . . . 26

2.11.Representation of MVBs and exosomes formation . . . 27

2.12.Schematic representation of exosomes size, compared to that of other biological particles[1] . . . 28

2.13. Structure of CD9 . . . 29

2.14.List of some cancerous disorders in which exosomes analysis can be used as a diagnostic tool . . . 30

2.15.Scheme showing many features concerning exosomes, their structure and their load . . . 31

3.1. Schematic representation of light coming from an ideal point in the object plane, collected by the objective lens . . . 33

3.2. Focal eld intensity prole along the lateral and axial optical coordi-nates . . . 34

3.3. PSF intensity distribution in a plane perpendicular and parallel and centered to the optical axis . . . 35

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List of Figures

3.6. Epiuorescence microscope setup . . . 39

3.7. Confocal microscope setup . . . 40

3.8. Comparison between confocal and excitation PSF along lateral and axial coordinates . . . 41

3.9. Confocal PSF in a plane perpendicular and parallel and centered to the optical axis . . . 41

3.10.Example of NHS-uorophore . . . 43

3.11.Atto633 excitation and emission spectra . . . 44

3.12.DiD structure . . . 45

3.13.Emission spectra of some lipophilic stains, including DiD . . . 45

3.14.Biotin structure . . . 46

3.15.Fluorescein-cadaverine structure . . . 47

3.16.Fluorescein excitation and emission spectra . . . 47

3.17.Phycoerythrin excitation and emission spectra . . . 48

3.18.Atomic Force Microscopy setup . . . 50

3.19.Typical DLS setup . . . 53

3.20.Schematic representation of a particle and its electrical double layer, planes and potentials . . . 55

3.21.Schematic representation of a dialysis setup . . . 57

4.1. Schematic spectrophotometer setup. . . 62

4.2. DLS analysis of an exosomes sample before ltering, after ltering and after staining with 3 µM DiD uorophore. . . 68

5.1. Example of MSD curve for the Brownian motion of a particle . . . . 77

5.2. Example of MSD curve for the conned motion of a particle . . . 78

5.3. NTA distributions of 50 nm virtual beads with and without drift cor-rection . . . 81

5.4. NTA analysis on 500nm beads without drift correction . . . 82

5.5. NTA analysis on 500nm beads with drift correction . . . 83

5.6. Normalized NTA analysis of 500nm beads with and without drift cor-rection . . . 83

5.7. Size distribution of a 100nm beads sample imaged by Olympus con-focal microscope . . . 84

5.8. Size distribution of a 100nm beads sample imaged by SP5 confocal microscope . . . 85

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List of Figures

5.9. Normalized size distribution of a 100nm beads sample imaged by SP5

and Olympus confocal microscopes . . . 85

5.10.DLS Intensity distribution of a 100 nm beads sample . . . 86

5.11.DLS Number distribution of a 100 nm beads sample . . . 87

5.12.Normalized DLS intensity and number distributions of a 100 nm beads sample . . . 87

5.13.Normalized DLS intensity distribution compared to Olympus and SP5 NTA distributions of a 100 nm beads sample . . . 88

5.14.Normalized DLS number distribution compared to Olympus and SP5 NTA distributions of a 100 nm beads sample. . . 89

6.1. TEM image of exosomes sample, negative staining . . . 90

6.2. TEM images of exosomes sample, negative staining . . . 91

6.3. TEM imaging of exosome sample, resin embedding . . . 92

6.4. AFM height image of the exosome sample . . . 93

6.5. AFM image of exosome sample, phase imaging . . . 94

6.6. AFM image of exosome sample, derivate representation . . . 95

6.7. DLS analysis of a clean exosomes sample and the same sample stained with DiD at dierent concentrations (intensity distribution) . . . 96

6.8. DLS analysis of a clean exosomes sample stained with DiD at dier-ent concdier-entrations (number distribution) . . . 97

6.9. NTA size distribution of 30 nanoparticles uorescent polystirene beads imaged by Olympus confocal microscope . . . 98

6.10.NTA size distribution of 30 uorescent polystirene beads imaged by SP5 confocal microscope . . . 98

6.11.Normalized NTA size distributions of 30 vesicles imaged by SP5 and Olympus, compared with DLS size distribution of the same sample . . 99

6.12.Example of 2-colors confocal microscopy analysis . . . 100

6.13.Example of three color acquisition performed with Olympus confocal microscope . . . 101

6.14.Example of processed three color acquisition performed with Olympus confocal microscope . . . 102

6.15.NTA analysis of the ExoDiD channel . . . 103

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List of Figures

6.19.Absorption spectra of AuNS (blue), Pep-K coated AuNS (red) and Peg-COOH coated AuNS (black) . . . 106 6.20.Zeta potential measurements of AuNS (red), Pep-K coated AuNS

(green) and Peg-COOH coated AuNS (blue) . . . 107 6.21.DLS analysis of Au, AuPc, AuPcSt and AuPcStBi . . . 109 6.22.UV-vis analysis of Au, AuPc, AuPcSt and AuPcStBi . . . 110 6.23.NTA size distributions of ExoDiD (blue) and AuK (red) signals for

the AuKaNAbExo sample . . . 113 6.24.NTA size distributions of ExoDiD and AuK signals for the

AuKaN-WAbExo sample . . . 114 6.25.NTA size distributions of ExoDiD and AuK signals for the

AuKaNX-AbExo sample . . . 114 6.26.AuKaNAbExo sample imaged by TEM microscopy . . . 115 6.27.AuKaNAbExo sample imaged by STEM microscopy . . . 116

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List of Figures

Introduction

This thesis work is a part of a research project focused on detection and char-acterization of exosomal biomarkers as a tool for a new tumoral screening assay. Earlier diagnosis of cancerous conditions allows the use of simpler and more e-cient treatments for dealing with the disease. In order to do so, a screening assay should be performed on the biggest possible number of patients, therefore it should be easily applicable and predictive. In recent years, exosomes have emerged as a potential tool for diagnosing tumoral conditions; exosomes are biological nanopar-ticles that are exocytated from their mother cells as intercellular communication vehicles, carrying material from a cell to another. It has been demonstrated that their membrane composition can be used for detecting the presence and type of a tumoral state, and the diagnostic assay can be performed with easily obtainable uids such as blood, plasma and urine.

The main goal of my work has been to nd a mean for conjugating metal nanopar-ticles to exosomes via antibodies, which are able to recognise exosomal membrane proteins, in order to derive the biochemical composition of the exosomes sample by mean of optical methods. In parallel, a nanoparticle tracking analysis computer program has been developed, in order to determine both bound and unbound species size distributions.

The organisation of this thesis work runs through seven chapters.

The rst chapter Purpose introduces the project in which this work is in-serted. The main purpose of ExoNanoDi, the project this work is part of, is to create a nanoparticle-based immunometric assay to detect and characterize exoso-mal biomarkers that are physiological responses to pathologic (mainly cancerous) conditions. A brief description of the project and the purpose of the thesis work is illustrated.

The second chapter, Nanoparticles and Exosomes, describes the biological and chemical background to understand the techniques and methods used in the fol-lowing chapters. The properties and structure of nanoparticles, as well as the basis of the chemical reactions involved, will be addressed.

In the following chapter, Microscopy and Spectroscopy Techniques, I recall some theoretical basis in optical microscopy and uorescence mechanisms, and how

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List of Figures

description of dynamic light scattering techniques, and the theory of Brownian motion is addressed.

In the fourth chapter, Materials & Methods, the instruments and techniques used for characterising and detecting the exosomes, as well as the parameters used for their analyses, will be described: these include the Zetasizer for dynamic light scattering and zeta potential measurements, the UV-vis spectrophotometer, electronic microscopes and confocal microscopes. Moreover, a description of the materials used in this project, from nanoparticles to coating polymers, to antibod-ies and uorescent probes, to exosomes, with details on their specications, will be presented, together with the experimental protocols.

In chapter 5, Nanoparticle Tracking Analysis, the theoretical basis and the de-scription of the NTA algorithm, together with its implementation for the analyses of the samples will be given. The calibration procedure is extensively addressed from the experiments with fake computer-generated tracks to uorescent beads, with particular attention to the use of the drift correction.

The discussion proceeds in the sixth chapter, Results & Discussion, in which the results of the experiments, from the characterization of the single particles (e.g. exosomes and nanoparticles) to the coating procedure, from the exosome-antibody conjugation to the nal nanoparticle-antibody-exosome system, will be presented together with the analyses of the control samples. The experimental results will be addressed step by step, from the characterization of the single particle (e.g. exosomes and nanoparticles), to the coating procedure, to the exosome-antibody conjugation and to the nal system nanoparticle-antibody-exosome , with the pre-sentation of the results compared to the control samples. We will show that the successful nanoparticle-exosome conjugation has been achieved and that it can provide a >90% pure nanoparticle-exosome sample. Furthermore, the NTA algo-rithm has been successful in determining the size of specic nanoparticles (both uorescent beads used as a test and labeled exosomes) imaged with a confocal mi-croscope. In the end, some conclusions from these results and further optimizations and perspectives that can be implemented in the future will be discussed.

The nal chapter Conclusions & Perspectives addresses further optimizations and future improvements that can be implemented to obtain better results (e.g. a higher purity) and to include other physical properties (e.g. magnetic nanoparti-cles, multifunctional coating et cetera).

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1. Purpose

1.1. Project

1.1.1. ExoNanoDi

This thesis work was conducted within the project ExoNanoDi at NEST (Na-tional Enterprise for nanoScience and nanoTechnology) of Scuola Normale Supe-riore (SNS) in Pisa (Italy). ExoNanoDi is a project funded by Regione Toscana (Bando Unico R& S 2012, POR CREO FESR 2007/2013) to Exosomics Siena SpA, and SNS collaborates to this project being a subcontractor of Exosomics Siena SpA. The purpose of this project is to implement a new diagnostic immuno-metric assay based on exosomal markers. The assay consists in the detection and characterization of biological markers expressed by exosomes (biological nanoparti-cles exocytated from their mother cell, with which they share part of the proteomic prole) and their isolation and quantication for diagnostic purposes. This goal is achieved through conjugation of noble metal nanoparticles to exosomes via specic antibodies and their analysis by spectroscopy and microscopy.

1.1.2. Thesis work

Within the project, the purpose of this thesis work was investigating several means for nanoparticle/antibody and nanoparticle/exosome conjugation, characterizing the system via microscopy and nanoparticle tracking analysis (NTA) in a

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multi-2. Nanoparticles and Exosomes

2.1.

Biochemistry background 2.1.1. Amino Acids and Proteins

Amino acids are a class of molecules which possess a carboxylic (COOH) group and an amino (NH2) group, both linked to a single carbon atom called α-carbon,

to which an aminoacid specic side chain or residue (R) is also attached[2]. At physiologial pH (pH 7.4), carboxylic and amino groups are deprotonated and pro-tonated, respectively, as shown in Figure 2.1.

Figure 2.1.: Amino acids general structure (1) and structure at pH 7.4 (2)[2]

Amino acids can link to each other by a covalent bond called peptide or amide bond; the product chain, called polypeptide or protein has an amino end (N-terminus) and a carboxylic end (C-(N-terminus) (see Figure 2.2).

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2. Nanoparticles and Exosomes

charge, hydrophobicity et cetera); the structure of the most common aminoacids is schematized in Figure 2.3.

Figure 2.2.: Peptide bond formation, the carboxylic end of the rst amino acids and the amino end of the second bind together expelling a water molecule (dehydration)[3]

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Figure 2.3.: Structure of common amino acids [4]

2.2. Chemical reactions

In the following I will show the main chemical reaction used in this thesis work: Peptide chemistry and gold-thiol bond.

Peptide Chemistry

Peptide chemistry is a group of fast and high-yield reactions involving amines and carboxylic acids in order to form covalent amide bonds, that are usually called peptide chemical reactions.

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is the EDC/NHS reaction[5] (EDC, N'-ethylcarbodiimide hydrochloride and NHS, N-hydroxysuccinimide), schematized in Fig. 2.5. In this reaction the amide bond is catalyzed by the reagents, although they do not become a part of the nal crosslink between the residues.

Figure 2.4.: EDC/NHS Reaction: the carboxyl group is activated with EDC/NHS (1), then the amide bond is formed between the carboxyl group and the primary amine (2). R is the target (e.g. a protein or a molecule) and R1 and R2 are the side groups of the reagents −CH2CH3 and

−(CH2)3N+H (CH3)2Cl

−_{respectively, which are used to guarantee the}

solubility in water [5] Gold-thiol reaction

Another important reaction is the gold-thiol bond formation[6, 7], which is a quasi-covalent bond (∼45 kcal/mol, while the covalent bond strength varies from 50 to 110 kcal/mol, approximately) that exists between the SH group (thiol) and a gold surface. The mechanism of such a strong interaction is not completely understood, but the reaction occurring is probably a chemisorption between the thiolate species (R) and gold

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to bind very close to each other, opening to the possibility of the formation of a self-assembled monolayer (SAM), which will be described below. A representation of the dependence of the strength of the bond along a gold surface is plotted in Figure 2.5.

Figure 2.5.: Schematic representation of the 2D distribution of the gold-thiol bond strength. The distribution is obtained from a simulation of a thiolated chain linked to a gold surface, the depressions correspond to the most favored binding sites (hollow sites) for the thiol group, and the peaks are the least favored sites. The x an y units refer to the spacing between two gold atoms on the surface[6]

2.3. Antibodies

Antibodies[8] (collectively called immunoglobulins, abbreviated as Ig) are pro-teins synthesized in mammals by a category of cells known as B cells and have a key role within the immunitary system for their recognition qualities toward a specic target, called antigen.

Mammals have ve classes of antibodies (IgA, IgD, IgE, IgG and IgM), each of which involved in dierent biological processes. For our purpose we will discuss only the structure of IgG class antibodies, which are used for their structural simplicity.

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sites located at the end of each arm of the Y (see Figure 2.6, so that they could bind to two antigens. The central region connected to the arms is called tail or Fc region.

This antibody consists in four polypeptidic chains, two light or L chains (about 220 aminoacids) and two heavy or H chains (about 440 aminoacids), stabilized by non covalent bonds and disulde bonds.

The nal part of every arm is constituted by a sequence of 110 aminoacids from the L chain and other 110 from the H chain, called the variable region, of which only the nal 5-10 aminoacids of every chain (called the hypervariable region) constitute the actual antigen-binding site, while the rest is referred to as constant region.

Figure 2.6.: Schematic antibody structure; the terminal amines and carboxilic acids for each chain are explicitely shown; it must be noticed, however, that other possible free amines or carboxilic acids can be present on the "surface of the antibodies, being them present on the side chains of some aminoacids. [8]

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Antibody-antigene conjugation

The antibody-antigene conjugation[8] is a reversible conjugation made possible by many non covalent bonds such as electrostatic, hydrogen, hydrofobic and Van der Waals bonds. The shape complementarity between the antigen-binding site and the antigen helps to make the bond stable and specic.

We can describe the reversible bonding reaction as: Ag + Ab ←→ AgAb

At the equilibrium, for as many antibodies bonding to their antigen we will have as many antigens leaving the antibody to which they were conjugated. The anity of an antibody with its antigen is described by the anity constant KA, dened

as

KA= _[Ag][Ab][AgAb]

where [AgAb] , [Ag]and [Ab] refer to the concentration of the antigen-antibody complex, the antigen and the antibody respectively. Usually the dissociation con-stant KD is addressed in literature: it is dened as the inverse of the association

constant, and is equivalent to the nal concentration of heterodimers when half the theoretical number of total heterodimers are dissociated.

2.4. Metallic Nanoparticles

Nanoparticles (Nps) are inorganic structures with dimensions below 1 μm, made of a few hundred up to a few thousand atoms. Metallic nanoparticles have been used recently for their peculiar interaction with light which stems from the exis-tence of a Localized Surface Plasmon Resonance (LSPR): the particles resonantly absorb and scatter light upon excitation of their surface-plasmon oscillation.

In particular, in this thesis work noble metal nanoparticles will be considered, for which the wavelengths of the light used to exploit the above mentioned optical properties are in the visible and near infrared range.

2.4.1. LSPR and optical properties

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occur-2. Nanoparticles and Exosomes

ring when a metal particle is irradiated by light: the electric eld of incident light at appropriate frequency induces a coherent oscillating behavior of the particle conduction electronic density which can be approximated as a dipole oscillation.

Figure 2.7.: Schematic representation of plasmon oscillation for a sphere due to the interaction with incident light[10]

Theory

We want to relate the dipole plasmon frequency of a metal nanoparticle to its dielectric constant, that can be measured for the bulk metal. We consider the interaction of light with a dielectric sphere that is much smaller than the wave-length of the incident light (e.g. λ ≈ 400nm is the wavelenght for blue light and d ≈ 30nm is the diameter of the nanoparticles addressed in this work, therefore λ d). Under these circumstances, the electric eld of the light can be taken to be constant and homogeneous in order to calculate the light matter interaction using electrostatics rather than electrodynamics (quasistatic approximation).

Denoting the electric eld (assuming it is along the x axis) of the incident wave by E0 = E0x, where x is a unit vector, we want to solve the LaPlace equation

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E = −∇ϕ

in order to determine the electromagnetic eld surrounding the particle. We as-sume that the particle is a perfect sphere with radius a and we apply two boundary conditions:

1. ϕis continuos at the sphere surface

2. The normal component of D = E to the surface is continous

Solving LaPlace equation with these boundaries leads to the following formulation for the electric eld

Eout = E0x − αE0 x r3 − 3x r5(xx + yy + zz) where α = s−0 s+2ma

3 _{is the polarizability of the sphere, where}

s and m are

the dielectric constants of the material and of the medium, respectively. We can easily notice that the rst term of the equation is the applied incident eld, while the second is the induced dipole eld and is proportional to the polarizability, which is strongly dependent on the dimension on the nanoparticle and its dielectric constant, which is, in turn, dependent on the incident wavelenght.

In conclusion, the formula above links the induced dipole eld to the inci-dent wavelength, dependending on the material (via the dielectric constant) and the dimensions of the nanosphere; in particular, a resonance is expected when Re(s) = 2s. A more complete solution involving Maxwell equations shows that

the sphere acts as a radiating damped dipole, therefore contributing to the ex-tinction of the incident light through Rayleigh scattering and absorption; usually, elastic scattering is predominant for the bigger NPs (above 10 nm for Au NPs).

The resonance frequency is determined by the type of metal (through the disper-sion of its complex dielectric function), and by the shape and size of the nanopar-ticle. For the smaller nanoparticles, other factors (such as the interplay between the electronic mean free path and the dimension of the nanoparticles, and quan-tum eects) have to be taken into consideration, while higher multipole modes can occur in case of larger (radius >100 nm) or non spherical particles. All these

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considerations complicate the simple treatment presented above. Nevertheless, for noble metals nanoparticles[11], the plasmon resonance frequency is usually in the visible range (about 530 nm for 30 nm gold spheres), allowing the analysis with visible light optical instruments like spectrophotometers (exploiting the extinction cross section) and microscopes (exploiting the Rayleigh scattering).

2.4.2. Gold Nanospheres - Synthesis

Gold nanospheres (AuNS) were synthesized at NEST laboratory using Zhong method, which is a bottom-up, seed-mediated procedure for the synthesis of monodis-perse gold nanoparticles. Bottom-up means that the synthesis process begins with a small sized population (small gold nanoparticles of ∼20 nm diameter) that is iteratively grown to the desired size; in this case the process is seed-mediated, which means that a smaller size species is used as a clustering nucleus. The nal product is then stabilized using acrylate as a solvent, providing a coating meant to separate the nanoparticle while maintaining their shape[12].

This methods provides monodisperse gold nanoparticles with a well-dened plas-monic absorption peak, the dimensions of which can be tuned by changing the concentration of gold added to the seeds (see Figure 2.8) .

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Figure 2.8.: TEM (Transmission Electron Microscopy) image of 30 nm gold nanoparticles sample[12]

2.4.3. Coating Polymers

In order to stabilize and to make functionalizable metallic nanoparticles, the native coating should be substituted by another one. In this work, we considered two types of coating: Polyethilene Glycol (Peg) based ones, and hexapeptidic ones. Polyethylene Glycol is a polymer, the repeating monomers of which are ethylene oxide, with structural formula

H − (O − CH2− CH2)_n− OH

It has been shown [13] that Peg is biocompatible so it can be eventually used in in vivo studies. Moreover, a coating with Peg is known to provide steric stabilization

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to nanoparticles in a colloid. Peg chains can have two functional ends, so that the polymer can be bound to the nanoparticle on one side while leaving a functional group (e.g. a carboxyl or amine group) on the other side. For our purposes, two types of Peg are addressed:

Carboxy-Peg-Thiol

HS − (CH2− CH2− O)n− CH2− CH2− COOH

Amine-Peg-Thiol

HS − (CH2− CH2− O)n− CH2− CH2− N H2

The thiol end (−SH) could be bound to gold via gold-thiol reaction, leaving the other group free for allowing EDC/NHS mediated formation of amide bond. Furthermore, the amine/carboxylic end of the peg maintains the stability of the coated nanoparticles by providing a net charge which contributes to separate them from each other.

For this thesis work also hexapeptides were considered as coating agents. These peptides were engineered at NEST laboratory[12] and can provide both carboxyl and amino functional groups on one side, and a thiol (present in the side chain of a cysteine) on the other.

The hexapeptide used for this thesis work is the K peptide (pep-K, Figure 2.9), with aminoacid sequence CLPFFK, where the four central aminocids have hy-drophobic side chains.

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Figure 2.9.: Schematic structure of pep-K hexapeptide. Aminoacids used are Cys-teine (C), Leucine (L), Proline (P), Phenylalanine (F) and Lysine (K) [12]

Due to the four hydrophobic aminoacids, two close hexapeptides will repel water and form a Self Assembled Monolayer (SAM, Figure 2.10); this peculiar property provides a complete and stable coating for the nanoparticles by keeping water and therefore also other possibly competing or degrading molecules in solution far from the thiolated metallic surface.

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Figure 2.10.: Schematic representation of hexapeptide coated nanoparticles. In the red circle, a scheme for hexapeptides forming a Self Assembled Mono-layer

2.5. Exosomes

2.5.1. Overview

Exosomes[1] are small vesicles generated by the fusion of multivesicular endo-somes with the plasma membrane. Exoendo-somes are the mediators to many physio-logical processes (e.g. immune response), but their origin is common and can be

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endosomal system, together with endocytic vesicles, early and late endosomes, and lysosomes. Their behavior (see Figure 2.11 follows two dierent paths: they either fuse with lysosome (this degradates their content) or with the plasma membrane, the latter resulting in exocytosis of the internal vesicles outside the cell. This vesicles are called exosomes, and their diameter ranges from 30 to 100 nm[14].

Figure 2.11.: Representation of MVBs and exosomes formation. (LE: Late Endo-somes, EE: Early EndoEndo-somes, MIIC: MHC class II enriched com-partments, PM: Plasma Membrane). MHC stands for Major Histo-compatibility Complex, a family of surface molecules which mediate interactions of leukocytes, or white blood cells, with other leukocytes or body cells[15]

The behavior of a normal cell consists in exchanging biological material with its environment, and this transport is mediated also by nanoparticles, like exosomes. In pathological states, such as in case of cancer, this mechanism results in an increased expulsion of specic proteins expressed on the surface of the exosomes and of exosomes themselves. Further studies [16] proved that a correlation exists between exosomes characteristics (e.g. concentration) and the developement of certain cancerous states, so detecting and characterizing exosomes could be a very useful tool to diagnose tumors, also in their early stages.

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In Figure 2.12[1] I show the main biological particles present at the nano- and micro-scale, from the smallest to the biggest one. It is possible to nd exosomes and viruses (range <100nm), microvesicles and bacteria (range 100nm - 1 micron) and apoptotic bodies, platelets and cells which are bigger than 1 micron. Microvesicles are structures surrounded by a phospholipidic bilayer, they are 1001,000 nm in diameter but the lower cuto remains to be established; their size range overlaps that of bacteria and are formed by regulated release by budding of the plasma membrane. Apoptotic bodies (15 micron in diameter) are released as blebs of cells undergoing apoptosis (a process of programmed cell death due to environmental factors) and may contain fragmented DNA; of similar size are platelets, blood cells with no nucleus, whose main function is to stop bleeding.

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2.5.2. Exosomal Biomarkers

Exosomes[17, 16] contain a wide array of molecules including miRNA, DNA, membrane proteins (like tetraspanins, proteins with four transmembrane domains, shown in Figure 2.13); their presence depends on various factors, including the cell type or origin and its local environment, as specied in the table in Figure 2.14 for exosomes important in pathological states.

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Figure 2.14.: List of some cancerous disorders in which exosomes analysis can be used as a diagnostic tool[16]

Two known panexosomal biomarkers are the CD9 and CD63 molecules, mem-brane proteins both belonging to the family of tetraspanin proteins; these are used for recognition of exosomes and as a marker for certain cancerous diseases (Figure 2.14).

Another protein of interest is the PSMA (Prostate-Specic Membrane Antigen)[18], a transmembrane protein expressed in all types of prostatic tissue (therefore present in prostate derived exosomes), and a useful diagnostic target for prostate cancer.

Another biomarker that drove the interest in exosome research is miRNA (micro RNA), small RNA sequences of 20-22 nucleotids that have been shown to be a powerful mean for diagnosing cancer and cardiac pathologies [16]. Figure 2.15 shows a summary of the main biological objects present on the surface and in the inside of exosomes.

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Figure 2.15.: Scheme showing many features concerning exosomes, their structure and their load[16]

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3. Microscopy and Spectroscopy

Techniques

In this chapter I will introduce the theory behind the microscopy and spec-troscopy tools used in this work, starting from the theory of optical microscopy.

In an optical transmission microscope[19] the sample is illuminated by visible light and the transmitted light is collected through the objective by a camera (or by human eyes), so the information is given by the dierences in absorption of light between the sample and the medium. The resolution in optical microscopy depends on the wavelength of the used light and on the numerical aperture NA of the objective (see next paragraph):

N A = n·sinα

where n is the refractive index of the medium where the nal part of the objec-tive is immersed (and, in most of the best congurations, where also the sample is immersed) and α is the semi-aperture angle of the cone of collected light (Fig-ure 3.1).

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3. Microscopy and Spectroscopy Techniques

Figure 3.1.: Schematic representation of light coming from an ideal point in the object plane, collected by the objective lens[20]

3.1.

Resolution and Point Spread Function

The Point Spread Function (PSF) of a lens (or an optical system in general) is the 3D light distribution obtained when imaging an innitely small point. This distribution is not concentrated in a single point because of the diraction of light; its dimensions is therefore proportional to the eective light wavelength in the immersion medium. Moreover, light emitted in a transmission or uorescence microscope extends over a full sphere, although the lens gathers only a small portion of it, and this clipping contributes to the blurring in the reconstructed image that leads to the PSF. The dimensions of the PSF are therefore linked also to the above mentioned numerical aperture (NA).

The diraction pattern can be calculated from diraction theory in order to obtain an analytical expression of the PSF. I will describe the case of an optical system that has cylindrical symmetry; we can introduce dimensionless coordinates named optical units (o.u.):

Lateral coordinate v = r · _λ2π

msinα = r ·

2π λN A

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3. Microscopy and Spectroscopy Techniques Axial coordinate u = z ·_λ2π msin 2_{α = z ·}2π λ N A2 n

where z is the distance from the geometrical focal point along the optical axis. The intensity distribution of the focal eld in the lateral and axial coordinates are: I(0, v) ∝ |2J1(v) v | 2 I(u, 0) ∝sin( u 4) u 4 2

where J1(v) is the rst-order Bessel function of the rst kind.

Figure 3.2.: Focal eld intensity prole along the lateral (a) and axial (b) optical coordinates[19]

The rst nodes of the distribution shown in Figure 3.2 can be found and calcu-lated in physical radial and axial coordinates

r0 = 0.61λ_{N A}

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Figure 3.3.: PSF intensity distribution in a plane perpendicular (left) and paral-lel and centered (right) to the optical axis; side lobes are enhanced by logarithmic contrast stretching[19]

Figure 3.4.: 3D intensity distribution of a PSF shown by isosurfaces at dierent relative intensities: 1 (a), 0.06 (b) and 0.04 (c)[19]

According to the Rayleigh criterion, the minimum distance at which two ideal point sources can be resolved is when the maximum of the PSF of the rst point matches the rst minimum of the PSF of the second point, therefore

r0 = 0.61λ_{N A}

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Since every ideal point of the real image will be imaged by an intensity distri-bution matching the PSF of the optical system, the whole image will be

I(X, Y, Z) =´_−∞+∞´_−∞+∞´_−∞+∞O(x, y, z) · P SF (x − x0, y − y0, z − z0)dx0dy0dz0 which can be written as

I(X, Y, Z) = O(X, Y, Z) ⊗ P SF (x, y, z)

where I and O represent the image and object functions, X,Y,Z and x,y,z are the spatial coordinates in image and object space, respectively, and⊗ is the

convolu-tion operator.

3.2. Fluorescence and UV-Vis spectroscopy and microscopy

3.2.1. Theoretical considerations

Every molecule can exist in a discrete number of energy states [21]; the ones we consider in this discussion are the nuclear (or vibrational) and electronic ones, the wave functions of which can be treated separately within the Born-Oppenheimer approximation (since the nuclei can be assumed static during electronic transi-tions).

The absorption of a photon with energy Eexc = ~ωexcgives the necessary energy

to the system for changing his electron density conguration from a lower to a higher energy state (e.g. from the ground state to a vibrational excited state of the rst electronic excited state), then fast non-radiative processes lower the energy of the molecule (to the vibrational ground state of the excited electronic state) and then the passage from the excited state to the ground state again can happen with the emission of light with energy Eem = ~ωem < ~ωexc, called uorescence

light. The uorescence mechanism is described by the Jablonski diagram shown in Figure 3.5.

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Figure 3.5.: Jablonski diagram: bold lines refer to electronic states, thin lines refer to vibrational states of the nuclei. S0 and S1 are singlet ground and rst

excited states, respectively, while T1 is the rst triplet state. Arrows

de-scribe some possible transition. Excitation, transition from the ground state (both electronic and vibrational) to a vibrational state of the elec-tronic rst excited state. Internal Conversion, fast non-radiative conversion to the vibrational ground state. Intersystem Crossing, coupling between S1and T1vibrational state, leading to an intersystem

crossing from S1to T1.Fluorescence, transition from the vibrational

ground state of the rst excited electronic state to a vibrational state of the electronic ground state with emission of light. Phosphorescence, transition from the vibrational ground state of the triplet electronic state to a vibrational state of the electronic ground state with emission of light [21]

Since the molecule can be excited from the ground state to any vibrational level, there's a wide range of photon energies that can lead to excitation.

Light absortion is described by the Beer-Lambert law

I I0 = 10

−ε(λ)Cx

where I and I0 are the intensity of light after and before entering the sample,

ε(λ) is called molar extintion coeent (the plotted values of extinction coecients versus excitation wavelength is called extinction spectrum), C is the concentration

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of the sample and x is the optical path of light through the sample.

UV-vis spectroscopy is an analysis performed in solution to evaluate the ab-sorbance of a sample in solution, dened by Beer-Lambert law as

A = log10(T ) = ε(λ)Cl

where l is the total optical path and T is the transmittance measured by the instrument, dened as T = I0

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3.2.2. Fluorescence Microscopy

Figure 3.6.: Epiuorescence microscope setup[22]

In uorescence microscopy [19]the contrast is given by uorescence, either aut-ouorescence of the sample to be analyzed or given by uorescent markers. In epiuorescence microscopy, the sample is illuminated with light that matches the uorophore excitation wavelength and the isotropically emitted uorescence light (with longer wavelength) is collected and discriminated from the excitation light by a dichroic mirror (see Figure 3.6) and the image is formed on the retina of the observer or on a multichannel detector (e.g. photographic lms or digital cameras). This set-up can be used for implementing various techniques.

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3.2.3. Confocal Microscopy

A confocal microscope is congured according to the setup shown in Figure 2.20

Figure 3.7.: Confocal microscope setup; it's important to note that the out of focus emitted light reaches only in minimal part the detector because of the spatial ltering made by the emission pinhole[23]

The laser light used for excitation (if necessary, spatially ltered through an excitation pinhole) is reected by a dichroic mirror and focused on the sample by the objective lens. Fluorescence light is then focused by the objective through the dichroic mirror to a detection pinhole, which selects only the light coming from the desired point which is then collected by the detection system. Since the light comes from a single point, to reconstruct a 2D plane a scanning system must be implemented: the two most common scanning methods are laser and specimen

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dierent formula for the PSF that accounts for both excitation and detection PSF and it's merely the product of the two

P SFconf(x, y, z) = P SFexc(x, y, z) · P SFdet(x, y, z)

As a result, the resulting PSF will have the side lobes substantially reduced and the main peak slightly narrowed, as shown in Figures 3.8 and 3.9.

Figure 3.8.: Comparison between confocal and excitation PSF along lateral (a) and axial (b) coordinates[19]

Figure 3.9.: Confocal PSF in a plane perpendicular (left) and parallel and centered (right) to the optical axis; side lobes are enhanced by logarithmic con-trast stretching [19]

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Therefore, the confocal PSF has virtually no side lobes.

Approximating the PSF in a plane as a Gaussian intensity distribution, we can obtain the lateral and axial resolution as its FWHM (Full Width at Half Maximum)

F W HMlateral,conf ≈ √1 1+β2 · λexc N A = 1 √ 1+β2 · F W HMlateral,exc F W HMaxial,conf ≈ √1 1+β2 · 2nλexc (N A)2 = 1 √ 1+β2 · F W HMaxial,exc where β = λexc λdet

Because of the dependence on β, the better choice for a uorescent probe would be the one with the shortest excitation wavelenght and the minimum Stoke's shift. However this is not always a feasible choice, since the shortest wavalegth (blue and UV) are generally more damaging for biological samples, moreover a minor Stoke's shift leads to a more dicult separation of excitation and emission light.

The main advantage of confocal microscopy is the enhancement in axial res-olution. Since the light coming from the out-of-focus planes is stopped by the detection pinhole, the signal from which the image is reconstructed comes mainly from the selected optical plane; on the contrary, the image provided by conven-tional optical microscopy is the resulting image of the collapse of all the specimen's optical planes into one, although the signals coming from out-of-focus parts are blurred.

3.2.4. Fluorescent Tools and Dyes

In order to visualize some parts of the samples in uorescence microscopy, the desired molecules or structures are usually bound to a uorophore; this procedure is called labeling or staining. The labeling procedure can be performed chemically (e.g. by using peptide chemistry), via antibody-antigen interaction (in this case it

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NHS-Fluorophores

Fluorophores are a class of uorophores which includes a functional NHS-activated carboxyl end. This NHS-end reacts with amine groups and it's already preactivated. In this work, ATTO633-NHS uorescent probes is used. ATTO633 is a uorophore synthesized by ATTO-tec, which displays a positive neat charge.

Figure 3.10.: Example of NHS-uorophore (ATTO495-NHS). It's possible to recog-nize the NHS-activated carboxylic end (bottom) connected to the uo-rescent molecule (top)[24]

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Figure 3.11.: Atto633 excitation and emission spectra[25]

Membrane Stains

A membrane stain used within this work is DiD, a uorophore of the class of long-chain dialkylcarbocyanines. This class displays a structure composed by a uorophore with two long aliphatic (alkyl) tales that interpose with phospholipids present in the lipidic or plasmatic double membrane. The spectral properties of the dialkylcarbocyanines are largely independent of the lengths of the alkyl chains, but are instead determined by the terminal ring systems and the length of the connecting bridge. They have extremely high extinction coecients and moderate uorescence quantum yields in a lipidic environment, while they display mild uorescence in non-lipidic environment.

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Figure 3.12.: DiD structure. It's possible to recognize the alkyl chains (bottom) con-nected to the uorescent molecule (top)[26]

Figure 3.13.: Emission spectra of some lipophilic stains, including DiD[27]

Biotinylated Fluorophores

Fluorophores can be bound to biotin, in order to use the biotin-(strept)avidin interaction.

Biotin (Figure 3.14) is a small vitamin (244 Da) which can be conjugated to specic proteins without signicantly altering their biological activity. The highly

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specic interaction of biotin-binding (like avidin) proteins with biotin make it a useful tool in assay systems designed to detect and target biological analytes.

Figure 3.14.: Biotin structure[28]

Avidin has a very high anity for up to four biotin molecules and is stable and functional over a wide range of pH and temperature. Avidin is useful for the detection and protein purication of biotinylated molecules in a variety of conditions. The specic interaction of avidin with biotin makes it a useful tool for detection systems. In this work I used a variant of the Avidin, called Streptavidin, which has a lower number of non-specic binding sites (e.g. glycosilation sites) than Avidin and it's slightly smaller (60 kDa instead of 68 kDa).

Fluorescein Cadaverine

Fluorescein Cadaverine structure (Figure 3.15) shows an amino functional group bound to a uorophore.

Fluorescein is a uorescent tracer commonly used in microscopy. In cellular bi-ology, the isothiocyanate derivative of uorescein is often used to label and track cells in uorescence microscopy applications (FITC, Fluorescein IsoThioCyanate). Cadaverine is a diamine compound with formula NH2(CH2)5N H2 produced by

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Figure 3.15.: Fluorescein-cadaverine structure[29]

Figure 3.16.: Fluorescein excitation and emission spectra[30]

Phycoerythrin

Phycoerythrin[31] (R-PE or simply PE) is a uorescent protein isolated from red algae that exhibits extremely bright red-orange uorescence with high quantum yields. It is excited by laser lines from 488 to 561 nm, with absorbance maxima at 496, 546, and 565 nm and a uorescence emission peak at 578 nm. It can be found commercially conjugated to a variety of primary antibodies, secondary antibodies, and streptavidin. Its excitation and emission spectra are plotted below.

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Figure 3.17.: Phycoerythrin excitation and emission spectra[32]

3.3. Transmission Electronic Microscopy

3.3.1. Theory

Transmission Electron Microscopy[33] uses elecron beams instead of visible photon beams. Since the de Broglie wavelength λdB of the electron can be written as

λdB = √_2mh

0eV

for an electron beam accelerated by a voltage V (m0 being the mass and e the

charge of the electron), and the resolution being proportional to the wavelength, we can see that TEM can reach resolutions of the order of the nanometer and less. The electron beam interacts with the electron density cloud around the atoms and scatters, creating a dierence in the transmission signal that is a function of the electron density. Other than conventional TEM microscopy, also the STEM (Scanning Transmission Electron Microscopy) analysis will be considered. The STEM works by scanning the specimen with a focused beam of electrons while

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contrast, in order to increase the electron density and preserving the biological sample, it is necessary to perform a xation and a staining procedures for both TEM and STEM.

3.3.2. Staining

Two main procedures for preparing samples for electron microscopy will be ad-dressed in this work: negative stainig and resin embedding.

Negative staining is a simple and rapid method to study the morphology of particulate specimens such as viruses, membranes and isolated proteins [34]. Neg-ative staining allows determination of shapes preserving the morphology of the structures, so it is particularly useful in exosomes characterization.

The specimen is stained with an electron-dense heavy metal solution which, drying, envelops the specimen. After the staining step, the sample will appear bright while the surrounding will appear dark due to the deposition of heavy metal atoms of the stain. The short time of treatment with heavy metal solution leads to the surface characterization but is not sucient for the penetration of the stain deeper inside the sample.

Resin embedding is a procedure consisting in xating the sample with a perme-ating resin in order to successively cut it in thin sections. Unlike negative staining, in resin embedding the staining procedure is longer (from 45 miuntes to one hour) so that the stain can enter the sample and reveal its inner ultrastructure.

3.4. Atomic Force Microscopy (AFM)

AFM scan [35] is performed moving a cantilever with a very thin and sharp tip, the position of which is measured by reecting a laser light onto a photodiode detector. While the tip moves up and down, following the dierent z-heigths of the sample during the scanning in the x-y plane, a three dimensional map of the surface of the sample is created. The relative movement of the sample in respect to the tip is achieved by positioning the sample on a piezelectric stage, which allows the sample to be moved by steps as small as 0.1 nm or even less.

The movement is done under computer control so that the tip won't push too hard the sample, damaging the surface of the tip itself. Uses of AFM other than

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surface scanning are not addressed, since they were not used in this work.

Figure 3.18.: Atomic Force Microscopy setup [35]

For sample preparation a mica support was used; mica is a group of sheet silicate (phyllosilicate) minerals including several closely related materials having close to perfect basal cleavage, meaning that are easy to split along denite crystallographic structural planes, resulting in a very smooth surface on which the sample can be hold. Other than simple height imaging, performed in contact mode (in which the instrument feedback is maintained constant), tapping mode has been used in this work: it maps topography by lightly tapping the surface with an oscillating probe tip. The cantilever's oscillation amplitude changes with sample surface topography. The main dierence between tapping and contact mode is that virtually eliminates lateral forces that can damage samples and reduce image resolution.

An important imaging mode used in this work is phase imaging: phase imaging consists in mapping the measured phase of the cantilever's periodic oscillations, in tapping mode, relative to the phase of the periodic signal that drives the cantilever. Changes in this measured phase often correspond to changes in physical properties across the sample surface.

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3.5.

Dynamic Light Scattering techniques

Dynamic light scattering techniques are used to investigate colloids, solutions con-taining nanoparticles with dimensions typically ranging from tenths of nanometers to few microns. They are typically based on the measuring the uctuation of light diused from these particles. In the most basic of these techniques (simply referred to as dynamic light scattering - DLS) the uctuations are ultimately caused by the Brownian motion, described in 3.5.1; in the laser doppler velocimetry technique (3.5.3) the uctuations are caused by the interference between two laser beams having undergone dierent doppler shift upon scattering by a moving particle. 3.5.1. Brownian motion

A small (few microns and below) particle in solution undergoes a random 3D motion due to the continuous random collisions with the molecules of the solvent; this stochastic motion is called Brownian motion.

In a purely diusive system [36] subjected to a stochastic external force (due to the random collisions of the particle with the molecules of the medium), the motion equation is

md_dt22x + µ

dx

dt = Fext

where µ is a costant, x is the position (in a unidimensional description) and m is the mass of the particle. Multiplying both parts of the equation for x and considering the mean over the ensemble, being < xFext >= 0 because of the

random nature of the external force, the equation becomes < mxd_dt2x2 > + < µx dx dt >=< xFext > < mxd_dt2x2 > +µ < x dx dt >= 0 < m(d[x( dx dt)] dt ) > − < m dx dt 2 > +µ < xdx_dt >= 0

The mean of the product of velocity and position doesn't change with time (due to the random nature of motion), so the rst term is zero

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3. Microscopy and Spectroscopy Techniques − < m dx dt 2 > +µ₂ d dt < x 2 _{>= 0} − < mv2 _{> +}µ 2 d dt < x 2 _{>= 0} d<x2_> dt = 2 <mv2_> µ = 2 kT µ

In the last passage the average over the ensemble of the kinetic energy is con-sidered equal to the thermal agitation 1

2kT. Considering all three dimensions and

integrating from 0 to t we obtain < R2 _{>= 6kT} t

µ

which is the mean square displacement of the particle after a time t.

If we consider the particles density over time and position ρ(x, t) and solving the diusion equation δρ

δt = D δ2_ρ

δx2 where D is the diusion coecient, we obtain a

Gaussian with second moment equal to < x2 >= 2Dt

=⇒< R2 >= 6Dt = 6kT_µt =⇒ D = kT_µ

Now we have a relation between the diusion coecient and µ, which quanties the viscous frictional force. From Einstein relation we can derive µ = 6πηr where η is the viscosity and r is the radius of the particle. Therefore

D = kT 6πηr

Concluding, if we're able to obtain the diusion coecient we can derive the size of the particle.

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(both AuNS and exosomes) in solution. A typical DLS setup is shown in Fig. 3.19.

Figure 3.19.: Typical DLS setup

The instrument used for performing DLS (Zetasizer, Malvern) calculates second order autocorrelation of the Rayleigh scattered light from the particles undergoing Brownian motion in the medium, in order to obtain the diusion coecient

g2(τ ) = <I(t)I(t+τ )>_<I(t)>2 = 1 + [g

1_{(τ )]}2

g2_{(τ )} _{is the second order autocorrelation functions, I (t) is the signal intensity}

and g1_{(τ )} _{is the rst order autocorrelation function, which describes}

autocorrela-tion of the amplitude of the electric eld given the delay τ. For spherical Brownian particles diusing in the solvent, it can be shown that the rst order correlation function decays exponentially

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with the decay rate given by ζ = q2_2D

q = 4πn_λ sin θ₂

Where q is the scattering wavevector, n is the refractive index of the sample, θ is the angle between the detector and the beam, λ is the wavelength used.

Reversing the equation we can derive the diusion coecient D hence the hy-drodynamic radius or diameter of the particle.

The output of this technique is actually a distribution for the hydrodynamic diameters weighted by the intensity of the diused light. In order to estimate the distribution weighted of the number of particles, the scattering cross-section must be considered. From the Mie theory we can write a relation between the scattering cross-section σ of a microvesicle with the diameter that is at least an order of magnitude less than the wavelength of the light used. For example, using a red laser (647 nm) the relation is true for particles of about 65 nm and less, so it's suitable for the smallest exosomes. The relation is

σ = d_λ64

m2₋₁

m2₊₂

2

where d is the particle diameter, λ is the wavelength of the used light and m = nv

nm

is the ratio between the refractive index of the vesiclencand of the medium nm. We

can see that increasing the diameter by an order of magnitude increases the cross section by six orders of magnitude, therefore the signal from the bigger particles can overwhelm the signal of the smaller ones in a polydisperse sample.

Furthermore, the measure gives the hydrodynamic radius of the particle, which is slightly bigger than the actual radius because of the clathrate, a cage-like structure composed by the molecules of the solvent that encircles the particle when put in a uid medium (e.g. water).

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two regions, an inner region, called the Stern layer, where the ions are strongly bound to the particle surface and outer region, called diusive layer, where the interaction is weaker. Ions within the Stern layer can be considered to move with the particle under Brownian motion, while the diusive layer does not. The boundary between these regions is called slipping plane, and the potential at the slipping plane is called Zeta potential.

Figure 3.20.: Schematic representation of a particle and its electrical double layer, planes and potentials

Zeta potential is used to give an indication of the stability of a colloidal solution (e.g. a nanoparticles solution) since it can be related to the surface charge of the colloid, therefore the higher the absolute value of the zeta potential, the higher the surface charge, the higher is the electrostatic repulsion among the particles.

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Zeta potential is related to the electrophoretic mobility UE

UE = _Ev

(where v is the velocity of the particle under the action of an applied electric eld E) by the Henry equation:

UE = 2εzf (ka)_3η

where UE is the electrophoretic mobility, z is the zeta potential, f(ka) is the

Henry function (aproximated to 1.5 for nanoparticles larger than 200 nm and to 1.0 for smaller particles, with k the reciprocal of the thickness of the double layer or Debye length and a the radius of the particle), ε is the dielectric constant and η is the viscosity of the medium.

Zeta potential can be obtained measuring the electrophoretic mobility of the sample by Laser Doppler Velocimetry (LDV) of the particles under the action of an electric eld, while ε, Henry function aproximation and η are given as input parameters.

3.6. Dialysis

Dialysis[37] is a technique which allows the separation of small sized species from bigger sized species present in the same (colloidal) solution by mean of a membrane with a pore size that allows the passage of only the smaller species.

The buer solution containing the sample is inserted inside the membrane, which is closed using appropriate clamps and then placed in a container lled with many orders of magnitudes higher amount of the desired buer solution (usually the same or a very similar buer to the one inside the membrane); the solution with the inserted lled membrane is stirred continuously to favor the diusion of the smaller species, which will cross the membrane because of the concentration gradient. As a result, the smaller species will be many order-of-magnitude-folds diluted in the sample while maintaining the species with bigger size inside the membrane.

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Figure 3.21.: Schematic representation of a dialysis setup, depicting the passage of small particles (empty dots) through the membrane. The white object on the bottom represents the magnetic stirrer, the black lled dot the species to be puried.[37]

Since the dialysis membrane consists of a spongy matrix of crosslinked polymers, the pore rating referred to as Molecular Weight Cut O (MWCO) is an indirect measure of the retention performance. More precisely, the membrane MWCO is determined as the solute size that is retained by at least 90%. However, since a solute's permeability is also dependent upon molecular shape, degree of hydration, ionic charge and polarity, it is recommended to select a MWCO that is half the size of the MW of the species to be retained and/or twice the size of the MW of the species intended to pass through.

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4. Materials & Methods

4.1. Various materials and methods

In this sections are listed basic biological and chemical materials and solutions (with their acronims) that will be cited in the following chapters.

Materials

Polyethylene Glycol: Peg polymers were purchased lyophilized from Rapp Polymere and will be used as a 2 mg/ml DMSO solution.

Hexapeptides: K-peptide has been synthesized at NEST and will be used as a 1 mg/ml DMF solution

NHS-Fluorophores: ATTO633-NHS Ester and Fluorescein-NHS Ester were purchased from ATTO-Tec and Sigma Aldrich respectively. Both will be used as a 100 mg/ml DMSO solution (10 µl aliquotes).

Fluorescein Cadaverine: Fluorescein Cadaverine was purchased lyophilized from Molecular Probes and will be used as a 1 mM DMSO solution.

Membrane stains: DiD stain was purchased by Life Technologies and will be used as a 1 mM DMSO solution.

Biotinylated Fluorophores: ATTO633-biotin was purchased from ATTO-Tec and will be used as 10 µl aliquotes (100 mg/ml DMSO solution)

Streptavidin: purchased lyophilized by Sigma Aldrich, 1 mg/ml PBS solution, 10 µl aliquotes.

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4. Materials & Methods

Dialysis capsule, Float-A-Lyzer, 1 MDa MWCO purchased from Spectra Por Biotech

Solutions

DMSO, Dimethyl sulfoxide

TEA, Triethanolamine

DMF, Dimethylformamide

PBS, Phosphate Buer Solution

Milli-Q, Ultrapure water

The details on most of the used instruments and techniques are given in the fol-lowing sections; here is the list of the standard laboratory supplies that don't need further discussion or details:

Centrifuge: Eppendorf Centrifuge 5415R with temperature control

Thermomixer: Eppendorf Thermomixer Comfort

Sonicator: Sonica Ultrasonic Cleaner 220ETH-S3

4.2. Zetasizer

DLS and Zeta Potential measurements were performed using a Malvern Nano ZS90.

4.2.1. DLS

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Dispersant: PBS solutions (obtained from ICN PBS Tablets following man-ufacturer instructions) or Water (with milli-Q solutions)

Temperature: 25° C

Equilibration Time: 180 s

Cell: ZEN 0117 - Disposable low volume cuvette (100 µl)

Measurement angle: 90°

Measurement number: 20

Runs: 50

Run duration: 25 s

Advanced: Automatic Positioning

Data processing: General Purpose

4.2.2. Zeta Potential

Material: Protein (Exosomes) or Gold (Nanoparticles)

Dispersant:PBS solutions (obtained from ICN PBS Tablets following manu-facturer instructions) or Water (with milli-Q or TEA solutions)

Temperature: 25° C

Equilibration Time: 180 s

Cell: Clear disposable Zeta cuvette

Measurement number: 20

Runs: Automatic

Run duration: Automatic

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4.3. UV-vis Spectroscopy

UV-vis spectroscopic measurements were performed using a Jasco V-550 spec-trophotometer.

This is a double beam spectrophotometer (see Figure 4.1) in which two cuvettes have to be inserted, the one with the sample and the one with the reference, which should be lled with the same buer as the actual sample. Two lamps provide the necessary incident light at dierent wavelengths ranges, a Tungsten Lamp (for wavelengths of 350-900 nm) and a Deuterium Lamp (for wavelengths of 250-350 nm); the light passes through a lter (if needed) and a monochromator that provide light with tunable wavelength range.

The light beam is then sent iteratively through the sample and the reference cuvettes and the resulting light is gathered by a photodiode; the data are then processed to obtain the absorbance values and the nal spectrum is computed.

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Figure 4.1.: Schematic spectrophotometer setup. A deuterium or a tungsten lamp provide light at dierent wavelengths, the light is then ltered and monochromatized and sent iteratively to the reference and the sample cuvettes, then the intensity of light is recorded at dierent wavelengths, processed to obtain absorbance from Beer-Lambert law and plotted in an absorbance spectrum[38]

Below will be described the typical parameters set for the experiments; these parameters have been used unless stated otherwise.

Parameters

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4. Materials & Methods Band width: 1 nm

4.4. Confocal Fluorescence Microscopy

Confocal microscopy experiments were performed with two microscopes, Olym-pus IX81 inverted microscope with uoview-1000 confocal module, and Leica TCS SP5 with resonant scanner on an inverted DM6000 microscope, both equipped with spectral detectors.

4.4.1. Olympus

Scan mode: XYT

XY Dimension: 256x256 pixel (35,2x35,2 µm)

T Dimension: 0.036 s per frame (fast, only suitable for up to two colors) or 0.65 s per frame (slow)

Number of frames: 450 (fast) or 50 (slow)

Objective Lens: UPLSAPO 60x Water

Numerical Aperture: 1.20

Zoom: 6x

Pinhole Aperture: 300 µm

Laser 1 wavelength: 488nm

Laser 1 Power setting: 3%

Laser 2 Power setting: 8.3%

Laser 3 Power setting: 20%

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4. Materials & Methods Channel 1 emission wavelength: 495-540 nm

Channel 2 emission wavelength: 540-546 nm

Channel 3 emission wavelength: BA 650 IF lter (>630nm)

The parameters used were the same for every experiment, PMT gains are not reported since they were specic for every experiment in order to optimize contrast. If less than three colors were needed, the unnecessary laser had been turned o. 4.4.2. SP5

SP5 internal instrumentation doesn't make use of dichroic mirrors and lters like Olympus, but their purpose is fullled by acousto-optical devices called AOTF (Acousto-Optical Transmission Filter) and AOBS (Acousto-Optical Beam Split-ters) and by a patented system based on moving mirrors for separating the emitted light dispersed by a prism on the dierent detectors.

Scan mode: XYT

Scan Speed: 8000 Hz

XY Dimension: 256x256 pixel (25.8x25.8 µm)

T Dimension: 0.019 s per frame, resonant scanner

Number of frames: 1500

Objective Lens: HCX PL FLUOTAR 100x Oil

Numerical Aperture: 1.30

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4. Materials & Methods Laser 1 power setting: 20%

Laser 2 wavelength: 563nm (Argon Laser line)

Laser 2 power setting: 30%

Laser 3 wavelength: 633nm (HeNe Laser line)

Laser 3 power setting: 20%

Channel 2 emission wavelength: 550-560 nm (with 488 laser, for PE)

Channel 2 emission wavelength: 553-573 nm (with 563 laser, for AuNS)

The parameters used were the same for every experiment if not stated otherwise, PMT gains are not reported since they were specic for every experiment in order to optimize contrast. If less than three channels were needed, the unnecessary laser had been turned o.

4.5. Transmission Electronic Microscopy

Trasmission Electronic Microscopy measurements were performed with a Carl Zeiss Libra 120 Plus microscope with an in-column Omega lter, used to select the range of electron energies in order to improve the contrast.

4.5.1. Negative Staining

A carbon coated grid (Cu 300 mesh, 3.05 mm wide) is placed upon a drop of the specimen solution and left adsorbing for 30 minutes. The sample is then drained by putting the grid on a sheet of drying paper at an angle of 45° and washed twice for 1 minute and once for 5 minutes, the washing procedure consists in posing the grid upon a drop of milli-Q water. The specimen is then stained with a 3% solution of uranyl acetate in 20% EtOH acqueous solution for 30 seconds, then it is drained and ready for the analysis.

Detection and characterization of exosomal biomarkers via noble metal nanoparticles conjugation