Magnetic nanostructured materials for advanced bio ...117509/FULLTEXT01.pdf · Functional Materials...

Magnetic nanostructured materials for

advanced bio-applications

Andrea Fornara

Licentiate Thesis

Stockholm 2008

Division of Functional Materials

Department of Microelectronics and Applied Physics

School of Information and Communication Technology

Royal Institute of Technology

Postal address Division of Functional Materials

Department of Microelectronics and

Applied Physics

Royal Institute of Technology

Electrum 229

Isafjordsgatan 22

SE 164 40, Kista, Sweden

Supervisor Mamoun Muhammed

Email: [email protected]

TRITA-ICT/MAP AVH Report 2008:15 ISSN 1653-7610 ISRN KTH/ICT-MAP/AVH-2008:15-SE ISBN 978-91-7415-184-8 © Andrea Fornara, 2008

Universitetsservice US AB, Stockholm 2008

Functional Materials Division, KTH, 2008 i

Abstract In the recent years, nanostructured magnetic materials and their use in biomedical and

biotechnological applications have received a lot of attention. In this thesis, we developed

tailored magnetic nanoparticles for advanced bio-applications, such as direct detection of

antibodies in biological samples and stimuli responsive drug delivery system.

For sensitive and selective detection of biomolecules, thermally blocked iron oxide

nanoparticles with specific magnetic properties are synthesized by thermal hydrolysis to

achieve a narrow size distribution just above the superparamagnetic limit. The prepared

nanoparticles were characterized and functionalized with biomolecules for use in a

successful biosensor system. We have demonstrated the applicability of this type of

nanoparticles for the detection of Brucella antibodies as model compound in serum

samples and very low detection limits were achieved (0.05 μg/mL).

The second part is concerning an in-depth investigation of the evolution of the thermally

blocked magnetic nanoparticles. In this study, the formation of the nanoparticles at

different stages during the synthesis was investigated by high resolution electron

microscopy and correlated to their magnetic properties. At early stage of the high

temperature synthesis, small nuclei of 3.5 nm are formed and the particles size increases

successively until they reach a size of 17-20 nm. The small particles first exhibit

superparamagnetic behavior at the early stage of synthesis and then transform to

thermally blocked behavior as their size increases and passes the superparamagnetic limit.

The last section of the Thesis is related to the development of novel drug delivery

system based on magnetically controlled release rate. The system consists of hydrogel of

Pluronic FP127 incorporating superparamagnetic iron oxide nanoparticles to form a

ferrogel. The sol to gel formation of the hydrogel could be tailored to be solid at body

temperature and thus have the ability to be injected inside living biological tissues.

In order to evaluate the drug loading and release, the hydrophobic drug indomethacin

was selected as a model compound. The drug could be loaded in the ferrogel owning to

the oil in water micellar structure. We have studied the release rate from the ferrogel in

the absence and presence of magnetic field. We have demonstrated that the drug release

rate can be significantly enhanced by use of external magnetic field decreasing the half

ii

time of the release to more than 50% (from 3200 to 1500 min) upon the application of the

external magnetic field.

This makes the developed ferrogel a very promising drug delivery system that does not

require surgical implant procedure for medical treatment and gives the possibility of

enhancing the rate of release by external magnetic field.

Functional Materials Division, KTH, 2008 iii

LIST OF PAPERS This thesis is based on following publications: 1. Andrea Fornara; Petter Johansson; Karolina Petersson; Stefan Gustafsson; Jian Qin;

Eva Olsson; Dag Ilver; Anatol Krozer; Mamoun Muhammed and Christer Johansson "Tailored magnetic nanoparticles for direct and sensitive detection of biomolecules in biological samples” Nano Letters 2008, 8 (10), 3423-3428

2. Stefan Gustafsson; Andrea Fornara; Karolina Petersson; Christer Johansson; Mamoun Muhammed and Eva Olsson "Evolution of structural and magnetic properties of magnetite nanoparticles for biomedical applications.” Manuscript

3. Jian Qin; Isaac Asempah; Sophie Laurent; Andrea Fornara and Mamoun Muhammed "Injectable Superparamagnetic Ferrogels for Controlled Release of Hydrophobic Drugs " Accepted for publication in Advanced Materials

Other work not included: 1. Shanghua Li, Jian Qin, Andrea Fornara, Muhammet Toprak, Mamoun Muhammed,

and Do Kyung Kim “Synthesis and Magnetic Properties of Bulk Transparent PMMA/Fe-oxide Nanocomposites” Submitted to Nanotechnology

2. Andrea Fornara; Laura Cattaneo; Stefano Bonetti; Jian Qin; Johan Åkermann and Mamoun Muhammed "Multilayered magnetic nanowires: fabrication, characterization and magnetic alignment", Manuscript

3. Fei Ye; Helen Vallhov; Jian Qin; Eva Daskalaki; Muhammet S. Toprak; Andrea Fornara; Susanne Gabrielsson; Annika Scheynius and Mamoun Muhammed “Synthesis and Effects of High Aspect Ratio Gold Nanorods on Human Antigen Presenting Dendritic Cells ", Manuscript

iv

Conference presentations 1. Fornara, Andrea; Mikhaylova, Maria; Prieto Astalan, Andrea; Johansson, Christer; Krozer, Anatol; Muhammed, Mamoun "Development of Thermally Blocked Nanoparticles for Biodiagnostic Applications" (poster) Life is Matter - Life Matters: Conversations in Bionanotechnology, Oct 25-27, 2005, Göteborg/Sweden 2. Fornara, Andrea; Mikhaylova, Maria; Prieto Astalan, Andrea; Johansson, Christer; Krozer, Anatol; Muhammed, Mamoun "Development of Thermally Blocked Nanoparticles" BioNanoMaT, Bioinspired Nanomaterials for Medicine and Technologies, Nov 23-24, 2005, Marl/Germany 3. Fornara, Andrea; Muhammed, Mamoun "Magnetic Nanoparticles for Biodiagnostics Applications" (oral) BIODIAGNOSTICS Workshop, November 28-29, 2005, Göteborg/Sweden 4. Fornara, Andrea; Qin, Jian; Muhammed, Mamoun "PLLA-PEG Nanospheres Encapsulating Monodisperse Iron Oxide Nanoparticles for Simultaneous Drug Delivery and MRI Visualization (poster)" 6th International Conference on the Scientific and Clinical Applications of Magnetic Carriers, May 17-20, 2006, Krems/Austria 5. Fornara, Andrea; Qin, Jian; Muhammed, Mamoun "3-G Multifunctional Nanoparticles for Simultaneous Drug Delivery and MRI Visualization (poster)" 8th International Conference on Nanostructured Materials, Aug 20-25, 2006, Bangalore/India 6. Marian, Carmen M.; Fornara, Andrea; Qin, Jian; Muhammed, Mamoun "Optimized Magnetic Nanoparticle for High Resolution Contrast in MRI (poster)" 8th International Conference on Nanostructured Materials, Aug 20-25, 2006, Bangalore/India 7. Fornara, Andrea; Li, Shanghua; Muhammed, Mamoun "Synthesis and characterization of copper sulfide nanoparticles" (poster) International Symposium on Inorganic Interfacial Engineering (IEE 2006), June 20-21, 2006, Stockholm/Sweden 8. Fornara, Andrea "Synthesis and functionalization of magnetic nanoparticle" (oral), BIODIAGNOSTICS Workshop, April 16, 2007, Berlin/Germany 9. Fornara, Andrea; Cattaneo, Laura; Muhammed, Mamoun "Multilayer magnetic nanowires: fabrication, Characterization and nanodevice application" (poster) 2nd Multifunctional Nanocomposites & Nanomaterials: International Conference & Exhibition, January 11-13, 2008, Sharm El Sheikh/Egypt 10. Fornara, Andrea; Nanoparticles for biomedical applications (oral) 3rd Meeting of Italian Researchers in Sweden, January 25, 2008, Embassy of Italy in Stockholm/Sweden 11. Fornara, Andrea; Cattaneo, Laura; Bonetti, Stefano; Qin, Jian: Åkermann, Johan; Muhammed, Mamoun "Fabrication and characterization of multisegment nanowires" (poster) 9th International Conference on Nanostructured Materials, June 2-6, 2008, Rio de Janeiro/Brazil

Functional Materials Division, KTH, 2008 v

Contributions of the author Paper 1. Planning and performing the experiments, characterization of the samples,

evaluation of the results and writing the manuscript.

Paper 2. Planning and performing experiments, performing part of the characterization of

the samples, evaluation of the results and writing parts of the manuscript.

Paper 3. Participate in developing the idea, performing parts of the experiments, writing

parts of the manuscript.

vi

ABBREVIATIONS AND SYMBOLS AAS atomic absorption spectroscopy AOT aerosol OT, sodium dioctylsulphosuccinate APTMS 3-aminopropyltrimethoxysilane CMC critical micelle concentration [mol/L] CT computed tomography CTAB cetyltrimethylammonium bromide Dc superparamagnetic critical size [m] DDS drug delivery system DLS dynamic light scattering E anisotropy energy [eV] FT-IR Fourier transform infrared H magnetic field [T] HRTEM high-resolution transmission electron microscopy kB Boltzmann constant [m2kgs-2K-1] LPS lipopolysaccharide LCST lower critical solution temperature M magnetization [Am2kg-1] MRI magnetic resonance imaging NMDEA N-methyl diethanolamine NP nanoparticle NIPAAm N-isopropylacrylamide PEG poly(ethylene glycol) PEO poly(ethylene oxide) PF127 Pluronic F127 PLLA poly(L,L-lactide) PNIPAAm poly(N-isopropylacrylamide) PPO poly(propylene oxide) PVA poly(vinyl alcohol) SDS sodium dodecylsulphate SEM scanning electron microscopy SPION superparamagnetic iron oxide nanoparticle SQUID superconducting quantum interference device TEM transmission electron microscopy TMAOH tetramethylammonium hydroxide TSC trisodium citrate UV-Vis ultraviolet-visible VSM vibrating sample magnetometer XRD X-ray diffraction σ standard deviation τ relaxation time of magnetic particle [s] τΒ Brownian relaxation time of magnetic particle [s] τΝ Néel relaxation time of magnetic particle [s] χ magnetic susceptibility [cm3g-1]

Functional Materials Division, KTH, 2008 vii

Table of contents

ABSTRACT ................................................................................................................................................... I

LIST OF PAPERS ...................................................................................................................................... III

TABLE OF CONTENTS .......................................................................................................................... VII

1 INTRODUCTION ................................................................................................................................ 1

1.1 OBJECTIVES ....................................................................................................................................... 1 1.2 OUTLINE ............................................................................................................................................ 3 1.3 NANOSCALE MAGNETIC MATERIALS ................................................................................................. 5

1.3.1 Nanoparticles ........................................................................................................................... 5 1.3.1.1 Magnetic nanoparticles synthesis ...................................................................................................... 5 1.3.1.2 Magnetic properties of magnetic nanoparticles ................................................................................. 8

1.4 BIOMEDICAL APPLICATIONS OF NANOSCALE MAGNETIC MATERIALS ............................................... 15 1.4.1 Biosensor applications ........................................................................................................... 15 1.4.2 Drug delivery systems ............................................................................................................ 17 1.4.3 Magnetic Resonance Imaging ................................................................................................ 19 1.4.4 Hyperthermia ......................................................................................................................... 21 1.4.5 Bio-manipulation ................................................................................................................... 22 1.4.6 Multifunctional nanoparticles in biomedicine ....................................................................... 24

2 EXPERIMENTAL ............................................................................................................................. 25

2.1 MAGNETIC NANOPARTICLES ............................................................................................................ 25 2.1.1 Synthesis of magnetic nanoparticles ...................................................................................... 25 2.1.2 Evolution study of magnetic nanoparticles ............................................................................ 26 2.1.3 Surface modification of magnetic nanoparticles ................................................................... 26

2.2 FERROGEL PREPARATION ................................................................................................................. 27 2.3 CHARACTERIZATION ........................................................................................................................ 28 2.4 MAGNETIC RELAXATION MEASUREMENTS ....................................................................................... 29 2.5 DRUG RELEASE RATE MEASUREMENTS ............................................................................................ 31

3 RESULTS AND DISCUSSION ........................................................................................................ 33

3.1 MAGNETIC NANOPARTICLES ............................................................................................................ 33 3.1.1 Morphology and structure studies ......................................................................................... 33 3.1.2 Evolution study ...................................................................................................................... 35 3.1.3 Magnetic study ....................................................................................................................... 38 3.1.4 Modification of the surface of nanoparticles ......................................................................... 41 3.1.5 Detection of biomolecules ...................................................................................................... 42

3.2 FERROGEL ........................................................................................................................................ 45 3.2.1 Structure study ....................................................................................................................... 45 3.2.2 Magnetic study ....................................................................................................................... 46 3.2.3 Drug release .......................................................................................................................... 47

4 CONCLUSIONS ................................................................................................................................ 49

FUTURE WORK ......................................................................................................................................... 50

ACKNOWLEDGEMENTS ........................................................................................................................ 51

REFERENCES ............................................................................................................................................ 55

Functional Materials Division, KTH, 2008 1

1 Introduction

Nanotechnology, a broad and interdisciplinary research field involving engineering,

chemistry, physiology, biology and more, has been growing exponentially in the past few

years, showing great potential for several technical and medical applications, such as early

detection, accurate diagnosis and personalized treatment of diseases.

Nanoscale materials and devices are typically smaller than several hundred nanometers

and are comparable to the size of large biological molecules such as enzymes, receptors,

and antibodies. With a size about 100 to 10000 times smaller than human cells, these

nanoscale devices can offer unprecedented interactions with biomolecules both on the

surface of and inside cells.1

The use of nanomaterials in biotechnology and medicine merges the fields of material

science, chemistry, physics and biology. Nanostructured materials, such as nanoparticles,

nanowires, nanotubes and thin films, provide a particularly useful platform for successful

development of wide-ranging therapeutic and diagnostic applications in the biomedical

area.2 Due to the tremendous development of this field of research worldwide, the term

“nanomedicine” was recently coined 3 , long after nanomaterials started to impact

biomedical research.

Magnetic nanoparticles, in particular, have been developed and optimized for

fundamental scientific interest but they have also found many biomedical applications

ranging from contrast agents in magnetic resonance imaging to biosensing applications.4

The unique properties and utility of nanoparticles arise from their peculiarities, including

high surface to volume ratio, large percentage of surface atoms compare to bulk materials

and the size of nanoparticles is comparable to the one of biomolecules such as proteins

and polynucleic acids.5

1.1 Objectives

In this thesis, we are aiming at developing tailored magnetic materials at the nanoscale

for advanced bio-applications, such as direct detection of antibodies in biological samples

and in advanced drug delivery systems.

2

For sensitive and selective detection of biomolecules, magnetic nanoparticles with

specific magnetic properties are developed and used in a novel detection method, which is

fast, easy to use and robust sensor. In-depth studies of the formation of such nanoparticles

are carried out by combining electron microscopy studies with magnetic properties at

different stages during the synthesis.

Besides the work on diagnostic tools, a novel drug delivery system based on

magnetically controlled drug release of drug is prepared and studied. The system is based

on the integration of superparamagnetic iron oxide nanoparticles and Pluronic F127 gels.

Hydrophobic drug indomethacin is loaded in the ferrogel owing to the oil-in-water

micellar structure. The characteristic sol-gel transformation property makes the ferrogel

an injectable drug carrier, which will not require surgical implant procedure for medical

treatment.


1.2 Outline

This thesis deals with development of tailored magnetic nanomaterials for different bio-

applications, such as early diagnosis of diseases and injectable drug delivery systems.

The common base of the different studies included is the engineering approach to tackle

a problem: first specific requirements about the problem are acquired, determining the

main objective that has to be reached. Then some possible strategies are proposed and the

most viable ones are chosen in order to guide the work, and after following these

guidelines scientific data are collected and the results are discussed in order to check

whether they fulfill the expected objectives and requirements. This is an iterative process

in order to develop and improve the strategies and the results step by step.

Section 1.3 gives an overview on different nanoscale magnetic materials, in particular

nanoparticles. Synthesis procedures, mainly by chemical routes, are reviewed and briefly

described. The magnetic properties of such nanoparticles are also explained, in terms of

superparamagnetic phenomena and relaxation behavior in suspensions.

Section 1.4 introduces the applications of magnetic nanomaterials in the biomedical

field, both from a front-edge research as well as from a clinical point of view.

Nanomaterials have been used to develop and improve the sensitivity and selectivity of

sensors to detect and analyze different biological molecules, such as proteins, plasmids,

antibodies, antigens, bacteria and peptides. Drug delivery systems have also been greatly

improved by nanostructuring existing materials or creating novel nanocomposites. Iron

oxide nanoparticles are currently used in clinical applications for magnetic resonance

imaging, enhancing the contrast of biological tissues and thus providing more accurate

and sensitive diagnosis for numerous diseases. Hyperthermia and bio-manipulation are

two other areas where nanoparticles have been employed to improve disease treatments,

mainly tumors, and in the field of regenerative medicine by magnetic control of stem cells

for tissue reparation. A very recent trend in the nanomedicine field is the development of

multifunctional nanoparticles that are able to achieve different tasks simultaneously, i.e.

drug delivery, magnetic targeting, imaging and sensing the biological environment.

Chapter 2 details the experimental procedures followed for the fabrication, surface

modification and characterization of nanoparticles. Section 2.1 deals with the

4

experimental work performed for the synthesis of two classes of magnetic iron oxide

nanoparticles with tailored magnetic properties. Surface modification has been performed

to functionalize pristine nanoparticles for specific biomolecules detection as well as to

form stable suspensions in aqueous media. The preparation of magnetic hydrogel based

on superparamagnetic nanoparticles and Pluronic F127 polymer is reported in Section 2.2.

All the materials were characterized by different techniques to evaluate the physico-

chemical properties, which are described in Section 2.3. Specific experiments have been

performed to measure the relaxation of nanoparticles suspended in a liquid to detect

antibodies as well as to understand the drug release behavior from the ferrogel, as

explained in Section 2.4 and 2.5.

Chapter 3 discusses the results for the synthesis and characterization of nanomaterials

at different processing stages. Section 3.1 deals with the results of nanoparticle synthesis.

Nanoparticles with an average size of ca 20 nm were obtained, as confirmed by electron

microscopy. The evolution studies regarding the formation mechanism of such

nanoparticles are also reported. High resolution TEM images show the crystal structure

and oriented alignment of nanoparticles at an early stage that leads to the final crystal

structure, morphology and size distribution. Section 3.1.3 summarizes the magnetic

measurements obtained with different techniques. The nanoparticle suspension shows

thermally blocked properties with high initial susceptibility. The evolution process was

also studies and analyzed from a magnetic point of view. SPION nanoparticles for drug

delivery system show instead distinct superparamagnetic properties. Section 3.1.4 and

3.1.5 contains the results of the surface functionalization studies and the detection of

anybodies both in biological samples as well as in solutions.

Section 3.2 includes the structural and magnetic evaluation of the prepared ferrogel

system using different techniques. In Section 3.2.3 the drug release rate under different

conditions is reported, underlying the possibility of control the release rate from the

ferrogel by applying an external magnetic field.


1.3 Nanoscale Magnetic Materials

1.3.1 Nanoparticles

In the past decade, the synthesis and use of magnetic nanoparticles has been intensively

studied not only for its fundamental scientific interest but also for many technological

applications. For example, magnetic nanoparticles were applied in different research

fields, such as sensor developments, magnetic resonance imaging in medicine, drug

delivery systems, hyperthermia, and magnetic separation.6

The vast interest in nanostructured particles mainly arises from the fact that these

structures possess novel physical and magnetic properties that differ from the one of bulk

materials. This is significant in the case of nanoparticles that have a large surface to

volume ration and the percentage of surface atoms is large, resulting in characteristic and

distinct physical properties.5

1.3.1.1 Magnetic nanoparticles synthesis

In the last decades, several methods for the preparation of magnetic nanoparticles in the

nanometer scale have been proposed and developed. The most common approaches used

are chemical/physical vapor deposition7, mechanical attrition8 and chemical routes from

solutions 9 . Among several advantages, chemical synthesis in solutions gives the

opportunity to precisely tailor the particle size, morphology, distribution, structure as well

as their magnetic properties. They can be prepares as stable colloidal suspensions in

aqueous or organic solutions. 10 Several types of magnetic nanoparticles have been

synthesized with a number of different compositions and phases, including iron oxides,

such as Fe3O4 (magnetite) and γ-Fe2O3 (maghemite),11 pure metals, such as Fe and Co,12

spinel-type ferromagnets, such as MgFe2O4, MnFe2O4, and CoFe2O4,13 as well as alloys,

such as CoPt3 and FePt.14

6

Several chemical approaches can be used for the preparation of magnetic nanoparticles,

precipitation, sol-gel, hydrothermal, microemulsion and thermal decomposition of organic

precursors. These techniques will be briefly described in the following section.

Iron oxide nanoparticles, Fe3O4 and Fe2O3 currently used in clinical investigations as

magnetic resonance imaging (MRI) contrast agents are predominantly synthesized by

aqueous co-precipitation process with coating layers.15,16 This hydrolytic process consists

of increasing the pH of the metal-ions solutions resulting in the precipitation of

nanoparticles with the desired composition. The size, shape, and composition of the

magnetic nanoparticles depends very much on the types of salts used (e.g. chlorides,

sulfates, nitrates), the Fe2+/Fe3+ ratio, the reaction temperature, the pH value and ionic

strength of the media.17 Other magnetic nanoparticles of ferrite structures, containing

cobalt, zinc and manganese, are also prepared by this method.18,19 Recently, significant

advances in preparing monodisperse magnetite nanoparticles, of different sizes, have been

reported by the use of organic additives as stabilization and/or reducing agents, such as

PVA, TMAOH, TSC. With this method, spherical nanoparticles with size in the

nanometer range can be obtained, but with rather large size distribution. This technique

allows preparing large quantities of nanoparticles in a single batch in a cost-effective

way.9

Another route to prepare magnetic nanoparticles, in particular oxides, is the sol-gel

method. It is a solution chemistry-based technique to synthesize pure, stoichiometric and

monodisperse oxides nanoparticles, starting from metal precursors that undergoes slow

hydrolysis and polycondensation reactions to form a colloidal system, called sol. The sol

evolves and leads to the formation of a network containing a liquid phase, called gel.20

Nanoparticles with large size, ranging from tens to hundreds of nanometers with relatively

narrow size distribution, can be obtained. This method can be performed at low

temperatures and is suitable for large scale production.21

A third route for nanomaterials preparation is the hydrothermal method operating at high

temperature. It has been used mainly for the synthesis of ferrites nanoparticles. This

strategy is based on a general phase transfer and separation mechanism occurring at the

interfaces of the liquid, solid, and solution phases present during the synthesis in aqueous

or organic systems at high temperature and pressure.22 Reduction under hydrothermal


conditions has been used to prepare monodisperse nanoparticles, using metal salts,

ethylene glycol, sodium acetate, and polyethylene glycol. Typically, the reaction mixture

is stirred to form a clear solution, then sealed in a Teflon-lined stainless-steel autoclave,

heated to and maintained at temperatures as high as 200-300 °C for several hours (72 h).23

The control of nanoparticle formation, size and distribution can be achieved by choosing

the appropriate mixture of solvents and varying parameters such as temperature, pressure

and reaction time. This process requires high pressures and elevated temperature for a

prolong period of time, and the scale-up is not easy.9

Another way to prepare nanoparticles is to carry out the reaction in a microemulsion

system. A microemulsion is a thermodynamically stable isotropic dispersion of two

immiscible liquids, where the microdomain of either or both liquids is stabilized by an

interfacial film of surfactant molecules.24 Depending on relative concentrations, surfactant

molecules self-assemble into a variety of structures in the solvent mixture, such as

micelles, bilayers or vesicles. Most commonly used structures in nanoparticle synthesis

are micelles, either as reverse (water-in-oil) or normal (oil-in-water) form. In both cases,

the dispersed phase consists of monodisperse droplets in the size range of 2–100 nm.25

This dispersed phase provides a confined environment for the synthesis and formation of

nanoparticles.10

Using the microemulsion technique, metallic cobalt, cobalt/platinum alloys, gold-coated

cobalt/platinum nanoparticles and spinel ferrites have been synthesized.26,27 Several kinds

of surfactants have been applied for the formation of such “nano-reactors” in which the

nanoparticle synthesis takes place, e.g. AOT, 28 CTAB, 29 SDS, 30 and polyethoxylates

(Tween).31 The main advantage of emulsion methods is the fact that particle size can be

easily controlled by changing the size of micelles and that the “nano-reactors” are isolated

from each other, limiting the particle growth.

Magnetic nanoparticles have been prepared also by thermal decomposition methods,

initially used for the synthesis of high-quality semiconductor nanocrystals and oxides.32,33

This process takes place in non-aqueous media at elevated temperatures. Several types of

magnetic nanoparticles with control over size and shape can be prepared.13 Monodisperse

magnetic nanocrystals with small size can essentially be synthesized through the thermal

decomposition of organometallic compounds in high-boiling organic solvents containing

8

stabilizing surfactants.34 The organometallic precursors include metal acetylacetonates,

[M(acac)n], (M=Fe, Mn, Co, Ni, Cr; n=2 or 3, acac=acetylacetonate), metal cupferronates

[MxCupx] (M=metal ion; Cup=N-nitrosophenylhydroxylamine, C6H5N(NO)O-), or

carbonyls.35 Fatty acids, oleic acid, oleylamine and hexadecylamine are often used as

surfactants.36 The ratios of the starting reagents including organometallic compounds,

surfactant, and solvent are decisive parameters for the control of the size and morphology

of magnetic nanoparticles prepared by this method. The reaction temperature, reaction

time, as well as aging period may also be crucial for the precise control of size and

morphology.37

Different types of magnetic nanoparticles, such as Co, Pt, and their alloys, have been

prepared by this method using different surfactants and capping agents.38 Iron oxide

nanoparticles can also be prepared via decomposition of iron pentacarbonyl or through

one-step decomposition of iron pentacarbonyl in the presence of a mild oxidant.39 In order

to avoid the use of highly toxic compounds in this synthetic route, a novel strategy has

been proposed based on the fact that the key intermediate during this process is the iron

oleate complex. This revolutionary idea leads to advanced synthesis procedure that

involves first the decomposition of the iron oleate complex to form the seeds that

subsequently grow into the nanoparticles. Further advanced methods were proposed for

synthesis of iron oxide starting from iron oleate, stereate, and laureate as precursors.40 A

large scale synthesis route for iron oxide nanoparticles based on decomposition of iron

oleate complex has been reported.13 Iron oxide particles with very a narrow size

distribution, controlled morphology and high crystallinity can be obtained via thermal

decomposition routes that have great potential for large scale production of high quality

magnetic nanoparticles.

1.3.1.2 Magnetic properties of magnetic nanoparticles

The classification of bulk materials with respect to magnetic properties is usually based

on their magnetic susceptibility (χ), defined as the ratio between the induced

magnetization (Μ) and the applied magnetic field (Η). In diamagnetic materials, magnetic

dipoles are oriented antiparallel to H resulting in small negative susceptibility (-10-6 to -


10-3) and the magnetization is not retained when the external field is removed.

Paramagnetic materials possess permanent magnetic dipoles that align parallel to H and

the susceptibility is positive and temperature dependent according to Curie law (at high

temperatures).

For ferro-, ferri- and antiferromagnetic materials collective magnetism is crucial due to

the fact that permanent magnetic dipoles show exchange interactions. This leads to a

definition of a critical temperature (Curie temperature for ferromagnetic materials and

Néel temperature for antiferromagnets) below which a spontaneous magnetization is

exhibited.

The susceptibilities of these materials depend on their atomic structures, temperature,

and the external field H. 41 In bulk materials and large magnetic particles there is a

multidomain structure, where regions of uniform magnetization are separated by domain

walls. If the particle size is decreased, there is a critical volume below which it costs more

energy to create a domain wall than to support the external magnetostatic energy (applied

field) of the single-domain state. Below this critical size, typically in the order of tens of

nanometers, materials that exhibit collective magnetism become a single magnetic domain.

This is observed for magnetic nanoparticles, and in particular for iron oxide systems

developed for bio-medical applications. For spherical magnetite particle the critical

diameter has been determined to be ca 120 nm.42 A single-domain particle is uniformly

magnetized with all the spins aligned in the same direction. The magnetization can be

reversed by spin rotation since there are no domain walls to move. This is the reason for

the very high coercivity observed in small nanoparticles.9

Magnetic single-domain nanoparticles possess another important property, the

anisotropy energy, which refers to the preference of the magnetization to lie along

particular directions (with respect to the crystallographic directions) within the

nanoparticles. These directions minimize the magnetic energy and are called anisotropy

directions or easy axes. The magnetic energy of a nanoparticle increases with the tilt angle

between the magnetization vector and the easy directions, as shown in Figure 1.1.

10

Figure 1.1 Evolution of the magnetic energy with the tilt angle between the easy axis and the magnetization direction.43

The anisotropy energy, represented as the variation of the amplitude of this curve, is the

product of the crystal volume times the anisotropy constant. The anisotropy energy is a

function of the tilt angle and the following expression is commonly used:

E = KV sin2θ (1.1)

where K is the effective uniaxial magnetocrystalline anisotropy constant per unit volume,

θ is the angle between the magnetization direction and the easy axis of the particle, and V

is the volume of the particle. The anisotropy energy is dependent on several contributions,

such as bulk magnetocrystalline anisotropy, shape of the crystal, anisotropy constant, and

dipolar interaction between neighboring nanoparticles.43

In the case of nanoparticles with an average diameter in the range of few tens of

nanometers, the anisotropy energy becomes comparable with the thermal energy kBT

where kB is the Boltzmann constant and T the absolute temperature.

According to Boltzmann distribution:

∫=

20

)sin())(-(exp

)sin())(-(exp )( π

θθθ

θθθ

θθd

TkE

dTk

E

df

B

B (1.2)

it is possible to calculate the probability f between angle θ and θθ d+ . Two different

situations can occur:

In the first case: kBT << KV: )(f θ is large around 0=θ . The magnetic moment is fixed

along the easy direction of magnetization. This means that the thermal energy is not high

enough to overcome the energy barrier KV that separates the two energetically equivalent


easy directions of magnetization. Nanoparticles that exhibit this condition are called

thermally blocked particles, and their magnetic moment is blocked along a specific

crystallographic direction (easy axis).

In the second case: kBT > KV: where the thermal energy, is high enough to move the

magnetic moments away from the easy axis and the magnetization is easily flipped. In this

situation the system behaves like a paramagnet, instead of atomic magnetic moments,

there is now a giant (super) moment inside each nanoparticle. This phenomenon is called

superparamagnetism, a term coined by Bean and Livingston.44

For superparamagnetic nanoparticles, the magnetization evolution with the external

magnetic field is proportional to the Langevin function that takes into account the

Boltzman distribution of the energy level corresponding to the possible orientations of the

particle magnetization moment. This function is commonly used to fit experimental

magnetization curves of nanoparticles and to determine the size of the crystals and its

specific magnetization. The characteristic features of superparamagnetic nanoparticles are

their response to a magnetic field, where there are no coercivity field, no remanent

magnetization and very high field saturation of the magnetization.43

A typical feature of superparamagnetic nanoparticles is the Néel relaxation time, τN, that

is the time constant of the return to equilibrium of the magnetization after perturbation. It

can be expressed as:

)(exp0 TkKV

BN ττ = (1.3)

where τ0 is the pre-exponential factor called characteristic relaxation time (dependent on

anisotropy energy), kB is the Boltzmann constant and T is the absolute temperature. This

time is characteristic of the internal relaxation of the magnetization of nanoparticles.

According to equation (1.3), the volume of nanoparticle and the temperature are two

critical parameters affecting the Néel relaxation time and therefore the superparamagnetic

or thermally blocked behavior of the system.

There is a size limit that divides the internal relaxation occurring in single domains in

two different time regimes, slow and fast relaxation compared to a characteristic

measurement time of the methods used to investigate the magnetic behavior of the system.

The superparamagnetic nanoparticles relax rapidly and do not show any magnetic

12

remanence or coercivity, while the thermally blocked single domains have longer

relaxation time and show both remanent magnetization and coercivity.45

A blocking volume VB and a blocking temperature TB can be defined44 in order to

distinguish the superparamagnetic single domains from the thermally blocked ones:

⎟⎟⎠

⎞⎜⎜⎝

⎛=

0

explnτt

KTkV B

B (1.4)

⎟⎟⎠

⎞⎜⎜⎝

⎛=

0

explnτt

k

KVT

B

B (1.5)

where texp is the characteristic experiment time of the measuring technique being used. It

is important to notice that the blocking limit is dependent of the experiment time, and it is

possible to obtain different values of VB and TB for different techniques. For instance,

vibrating sample magnetometer (VSM) has a characteristic measurement time of 10 s,

while superconducting quantum interference device (SQUID) has a measuring time of ca

10-2 s and Mössbauer spectroscopy 10-8 s.46

When nanoparticles are suspended in a liquid, the Brownian motion of such

nanoparticles plays an important role. This physical phenomenon occurs due to

continuous collision between particles and the thermally activated liquid molecules, and

the effects are more pronounced for small particles in the sub-micron range. The

Brownian motion mechanism can be arbitrarily divided into a translational (linear) motion

and a rotational motion. The linear movement can be measured by dynamic light

scattering or photon correlation spectroscopy and can be described by the Stokes-Einstein

equation45:

23

TqkD

B

HL

πητ = (1.6)

where η is the viscosity of the liquid, DH the hydrodynamic diameter of the particle and q

is the light scattering wave number. The rotational motion can be described by a

characteristic rotational diffusion time, according to:

TkD

B

HB 2

3πητ = (1.7)


It is possible to determine the rotational Brownian relaxation of magnetic nanoparticles

through magnetic measurements of the frequency dependent complex susceptibility.47

For magnetic nanoparticles suspended in a liquid, an effective relaxation time τeff related

to magnetic measurements can be defined as follow:

BNeff τττ111

+= (1.8)

Both the Néel and the Brownian relaxation processes are contributing to this effective

time, while the translational motion time is neglected because it does not affect magnetic

measurements. Figure 1.2 represent the different time constants related to the different

relaxation mechanism for nanoparticle in suspension as a function of their radius.

Figure 1.2 Time constants vs. particle size for magnetite particles.48

The plot illustrated in Figure 1.2 is given for magnetite nanoparticles at room

temperature. The blocking radius is estimated as ~ 7 nm.48 There are in principle two

ways to measure the relaxation phenomena: either in the time domain or in the frequency

domain.

Dynamic measurements of the magnetic susceptibility can be used to measure the

relaxation time of nanoparticle suspension and estimate if they undergo Brownian of Néel

relaxation.45 The AC-susceptibility measurement determines the resonance frequency of

the nanoparticles suspended in the liquid.

14

The magnetization of a particle system in an alternating external magnetic field is given

by:

H)j(HM ''' χχχ −== (1.9)

where

( )( ) ∞+

∞ −+

−= χ

τπχχ

χ β10

21 fjf (1.10)

and M is the magnetization, H alternating (time dependent) external magnetic field,

χ frequency dependent complex magnetic susceptibility, τ is the characteristic relaxation

time for magnetic relaxation that particles undergo.

By measuring the complex susceptibility it is therefore possible to determine the

Brownian relaxation time and the mean hydrodynamic volume of the nanoparticles.


1.4 Biomedical applications of nanoscale magnetic materials

Over time, the focus of nanotechnology research has gradually shifted from

nanomaterials and investigation of their physicochemical properties to the use of these

properties in several applications. One of the fields that can enormously benefit from the

advancement in nanotechnology is biomedical research. In particular, highly specific

medical interventions at the nanoscale for curing disease and repairing damaged tissues

such as bones, muscles or nerves are emerging as nanomedicine area.3

Nanomaterials have a great impact on biomedical research especially in the last few

years: superparamagnetic iron oxide nanoparticles as MRI contrast agent were studied and

today these are commercial products49. Quantum Dots were also developed recently and

are currently use in biology and medicine50. The great advantages of nanomaterials in the

biomedical research field lies in its ability to operate on the same small scale as all the

intimate biochemical functions involved in the growth, development and ageing of the

human body.51 Only in a few cases a particular nanostructured material was developed

completely for a specific biomedical application. More frequently, nanomaterials

discovered in the past are now further developed and applied in different biomedical

fields.3

Still there are a lot of challenges for the use of nanoparticles in medical applications.52

One of the main issues is certainly related to long-term safety of nanomaterials, both

developed for in vitro and in vivo applications. Not only toxicology tests have to be

developed specifically for nanomaterials, but also risk assessment and management have

to be defined.53,54

1.4.1 Biosensor applications

In the last few years huge efforts were directed towards the development of diagnostic

tools in order to improve the sensitivity of existing diagnostic techniques and to

considerably reduce the time and labor required for analysis. Many biosensors take hours

or even days and several successive steps and procedures are required to produce results,

16

so there is a clear need for devices that operate on a short timescale.55 Such devices could

have a major impact on the diagnosis of several diseases by allowing at-risk patients to

check tell-tale signs of proteins or other biomolecules by simply testing a small droplet of

blood or serum.56

A combination of magnetic nanoparticles and ultrasensitive magnetic field sensors is

nowadays showing a great potential as a perfect team to achieve these goals. In these

biosensors, the nanoparticles are coated or functionalized with chemical groups or entities

that will bind to the biomolecule that need to be detected. 57 In the case of sensors on a

substrate, these nanoparticle-biomolecule complexes then react with “probes” molecules

that are fixed onto the surface of the magnetic sensor. The presence of the magnetic

nanoparticles on the surface produces an output signal. In the case of label-free biosensors,

the nanoparticle-biomolecule complexes are directly detected by probing changes in

magnetic properties of the nanoparticles after the binding events. These kind of label-free

biosensors are extremely promising, especially for point of care applications, where the

assay should be simple, requiring no or minimum preparation. The possibility of analytes

detection directly in biological samples will lead to more economic, simple to use,

versatile and flexible sensors.58

A variety of diagnostic tests have been developed for the detection and quantification of

biological molecules, e.g. antibodies, in different biological fluids. Traditional

immunoassays depend on the use of radioactive, fluorescent or enzyme labeled-antibodies

as analytical tools to monitor biomolecular events.59 The enzyme-linked immunosorbent

assay (ELISA) is the most widely used for the detection of antibody-antigen

interactions60, both in routine diagnostics and research. These kinds of assays have a main

limitation due to the need of time consuming pre-treatment required for biological

samples preparation prior to analyses.61,62,63

Superparamagnetic iron oxide nanoparticles are used in several biomedical

applications64, including the quantification of biomolecular targets in cell lysates and

tissue extracts 65. They have also been used to detect larger biological entities, such as

bacteria, in solutions.66,67 In most cases, commercial beads containing magnetic multi-

cores with different surface layers have been used68, but these detection systems have


poor sensitivity and limited detection range due to a wide bead and magnetic domain size

distribution.

To overcome these limitations, we applied a different strategy for sensitive detection of

biomolecules in biological fluids. The sensing principle is based on susceptibility

measurements in an alternating (AC) magnetic field of the Brownian relaxation of

functionalized magnetic nanoparticles. 69 AC susceptibility is an excellent detection

technique, since it is simple, direct, and can be carried out in a compact device equipped

with a simplified readout system. Recently, the detection of specific DNA strands after

rolling circle amplification with commercial magnetic beads has been carried out

measuring large changes in Brownian relaxation.70

1.4.2 Drug delivery systems

Recent research on biocompatible materials has lead to a great development of a specific

field of medical devices engineering: the controlled drug delivery systems (DDS). DDS

can be specifically designed to control the release rate and/or amount of therapeutic

agents that will allow desired effects on target sites. Controlled drug release can be

achieved by combination of carrier materials and active agents.21 There are a few classes

of biocompatible materials used as carrier matrix for DDS, and these include solid lipid

nanoparticles,71,72,73 inorganic materials74,75 spheres76 and hydrogel systems77 fabricated

from biodegradable polymers.

Figure 1.3 Controlled drug delivery versus immediate release with repeated administration78

Repeated administration

18

In engineering drug release systems, the most important function of drug carriers is

regulating the drug release rate. Reducing the frequency of drug intake enables patients to

comply with dosing instructions. Most of the drug contents tend to be released rapidly

after the administration, which may cause rapid increase of the drug concentration in the

body. Repeated drug administration might lead to periods of ineffectiveness and toxicity

during medical treatment (Figure 1.3).

Drug delivery systems can improve several crucial properties of “free” drugs, such as

solubility, in vivo stability, pharmacokinetics, and biodistribution, enhancing their

efficacy and reducing side effects of the therapeutic compounds.79 Thus, engineered DDS

can prevent and considerably reduce risks related to underdosing and overdosing of

therapeutic drugs in patients as well as decrease the risks of side effects.80

There are several mechanisms used in the recent years to control the release rate of a

drug. Most commonly, controlled DDS is generally the diffusion- and dissolution-based

release system applicable to the release of drugs intended for the circulation or the

localization on the site.81 Recently, other mechanisms were also studied and developed in

order to have a detailed and tuned release of active compound within specific tissues and

cells, leading to so-called stimuli responsive DDS.5 Cationic lipids are used to cover the

surface of nanoparticles and promote cellular uptake and thus drug delivery inside specific

cells.82

Mesoporous silica pores can also act as stimuli responsive DDS when the caps of the

pore are removed by thiols compounds. 83 pH-responsive nanomaterials provide an

alternate mechanism for release, relying on the acidic conditions inside tumor and

inflamed tissues, cellular compartments including endosomes and lysosomes.84 Towards

this scope, magnetic nanoparticles were encapsulated in thermosensitive polymers for

controlled triggering of drug release. 85 Other thermosensitive DDS were recently

developed as thermosensitive nanoparticles based on PNIPAAm, a polymer which

undergoes structural changes at a certain temperature corresponding to the LCST.86,87

Nanoparticles can also provide effective carriers for large biomolecules such as DNA,

RNA, or proteins, protecting these biological entities from degradation and transporting

them across the cell-membrane barrier. The success of “safe” delivery of these


biomolecules provides access to gene therapy as well as protein-based therapeutic

approaches.5

Another strategy recently developed in the field of DDS is to engineer nanostructured

materials so that the release of drug would be triggered by the chemical environment.88

For example, microgel responsive to the concentration of glucose has been developed for

glucose-sensitive DDS.89 Hydrogel based on poly(aspartic acid) and poly(acrylic acid) are

demonstrated to be able to swell and shrink due to the change of salt concentration.90

Magnetic stimulation has also been used as activating mechanism for the release of

dopamine from nucleus accumbens shell of morphine-sensitized rats during abstinence.91

DC magnetic field has also been used to control the release of drug from an hydrogel

matrix in a “on-off” manner.92 More recently, hydrogel constructed with thermosensitive

polymer and magnetic NPs has been prepared and such system is of great promise in drug

delivery applications due to the dual stimuli-sensitivity.93 Hydrogel materials were also

reduced to nanoscale, developing “nanohydrogels” for drug delivery that can easily evade

the macrophage clearance with no opsonization or toxic effects.94

Still there is a need to improve the hydrogel systems to be able to have injectable

materials carrying a drug directly in the body without the need of surgical implantations.

It is advantageous to have a drug delivery system responsive to external signals, such as

magnetic field, so that the release rate could be carefully controlled to avoid underdosing

and overdosing of the therapeutic compounds. Part of this thesis deals with the

development of such novel ferrogel systems combing external stimuli response with the

possibility of injecting ferrogel in a liquid state. There are no reports in to-date literature

on such a system before.

1.4.3 Magnetic Resonance Imaging

Among a number of imaging techniques, such as optical imaging, ultrasound imaging,

positron emission tomography and X-ray tomography, Magnetic Resonance Imaging

(MRI) is one of the most powerful non-invasive imaging modalities utilized both in

biomedical research and clinical medicine today. 95 , 96 The current development of

magnetic nanomaterials, in particular nanoparticles, advances bio-imaging technologies in

20

terms of sensitivity, special resolution and other critical parameters.5 MRI imaging is

based on the property that hydrogen protons will align and process around an applied

magnetic field, B0. Upon application of a transverse radiofrequency (rf) pulse, these

protons are perturbed from B0. Then a relaxation phenomenon takes place whre these

protons return to their original state from the perturbed state. Two independent processes,

called longitudinal relaxation (T1-recovery) and transverse relaxation (T2-decay), can be

monitored to generate an MRI image. Local variation in relaxation, corresponding to

image contrast, arises from proton density as well as the chemical and physical nature of

the tissue under investigation.15

To obtain better images with well-defined mapping, contrast agents are utilized during

the imaging recording procedure. The main effect is to decrease relaxation times, either

T1 and/or T2, depending on the nature of the contrast agent used.97 Normally, gadolinium

and manganese chelates are clinically used to modify T1 relaxation98, producing brighter

images, while superparamagnetic iron oxide nanoparticles are used as T2 contrast agent,

giving a darker contrast in the tissue where they are accumulated.99

The effectiveness of a contrast agent can be described by its relaxivity, which is the

proportionality constant of the measured rate of relaxation, or R1 (1/T1) and R2 (1/T2),

over a range of contrast agent concentrations. 15,100

In the recent years, a lot of effort has been directed towards the development of

magnetic nanoparticles as MRI contrast agents. The most common strategy consist of

having iron oxide nanoparticles as magnetic core with tailored magnetic properties and

size distribution, and different surface functionalization to increase the stability of the

suspension in biological fluids and to target specific tissues of the body.97 Biocompatible

polymers, inorganic materials such as silica and gold, small pharmacophores such as

peptides, small organic ligands and proteins are the main classes of compounds used to

coat and functionalize the magnetic core of MRI contrast agent nanoparticles.43

To illustrate the wide interest of iron oxide nanoparticles in MRI imaging, a few

examples are reported here. Cancer research has received great benefits from MRI

techniques and magnetic nanoparticles have been extensively developed to improve the

detection, diagnosis and therapeutic management of solid tumors. Superparamagnetic iron

oxide nanoparticles (SPION) are currently used for clinical imaging of liver tumors and


prostate, breast and colon cancers as well as for the delineation of brain tumor volumes

and boundaries. 101 Next generation of active targeting based on nanoparticles has a

potential to offer significantly improved tumor detection and localization by exploiting

the unique molecular signatures of these diseases.102,103 Other applications of magnetic

nanoparticles enhanced MRI are in the field of cardiovascular medicine, including

myocardial injury, atherosclerosis and other vascular diseases.15

Molecular imaging, defines as the non-invasive in vivo visual representation,

characterization, and quantification of biological processes at the cellular and molecular

levels, has also been greatly affected by nanoparticles development. For instance,

molecular imaging allows sensitive and specific monitoring of key molecular targets and

host responses associated with early events in carcinogenesis and other diseases.104

Targeting of the inflammatory endothelial tissues was successfully performed with

superparamagnetic contrast agent both in vitro and in vivo models of inflammation105

Another successful application is specific tracking of cells in vivo after loading them with

magnetic nanoparticles functionalized with peptides and transfection agents.43,106 Novel

applications of nanoparticles and MRI in molecular imaging are related to studies of cell

migration and trafficking, apoptosis detection and imaging of enzyme activities.107

1.4.4 Hyperthermia

In the numerous applications of nanomaterials in medicine, cancer treatments with

nanoparticles have received great attention instead of traditional chemical therapy108. In

the last few years, a lot of studies have been conducted on magnetic nanoparticles that

generate heat when an alternating magnetic field is applied. This phenomenon is called

hyperthermia and it is extremely dependent on the size distribution of nanoparticles as

well as their magnetic properties. 109 In most studies, superparamagnetic nanocrystals

suspensions are tested in order to check the ability of energy absorption from an

oscillating magnetic field and its conversion into heat. This property can be used in vivo to

increase the temperature of tumor tissue and to destroy the pathological cells by

hyperthermia, since tumor cells are more sensitive to temperature increase than healthy

22

ones.43 The main parameter determining the heating of the tissue is the specific absorption

rate (SAR), defined as the rate at which electromagnetic energy is absorbed by a unit mass

of a biological material. For magnetic nanoparticles with superparamagnetic properties,

the SAR is precisely determined by the volume ratio of nanoparticles in the tissue and was

theoretically explained.110

Recent strategies for hyperthermia treatment are focusing in delivering and

accumulating magnetic nanoparticles in specific tissue.43 This has been proved in vitro by

selective remote inactivation of cancer cells by an AC magnetic field and evaluation of

the feasibility and survival benefit of this new hyperthermia approach is in progress on

animals, and first clinical trials have been started recently. 111 Superparamagnetic

nanoparticles are seen as a very promising agent for hyperthermia treatments, especially

in the form of multifunctional drug delivery system, but this new field of application

requires great reproducibility, control of size and magnetic properties during the synthesis

of nanomaterials.43

1.4.5 Bio-manipulation

In the recent years, a lot of effort has been put in the field of manipulation and remote

control of specific cellular components in vitro, and more importantly, in vivo, giving

clinicians and scientists a powerful tool for investigating cell function and molecular

signaling pathways.112 Furthermore, microparticles and nanoparticles have been used in

the field of biotechnology for different applications, including protein separation and

purification, protein detection and analysis, DNA extraction and biocatalytic

transformations.113,114,115

Magnetic separation techniques have several advantages in comparison to traditional

separation procedures: the process is very simple and all steps of the purification can take

place in one test tube without expensive liquid chromatography systems.116 Magnetic

materials in nanoscale dimensions have been widely used for affinity isolation of

proteins117, for capture and detection of bacteria at low concentration118 and for separation

of proteins from biological samples119.


Magnetic nanomaterials are useful tools for investigation of cell functions and molecular

signaling pathways, giving the possibility of probing ion channel activity, apoptosis

mechanisms, protein production and stress response. The actuation of magnetic particles

provides several advantages over other techniques for remote cell manipulation, such as

optical tweezers, nanoindentation and nanopatterned arrays. The main advantage is that

the process allows remote control of the nanoparticles at a distance, giving the possibility

of using this technique for future in-vivo applications.112 Magnetic nanomaterials, in

particular nanoparticles attached to membrane receptors, have been used since more than

a decade to investigate properties of the cytoplasm under an applied stress.120 Recently, by

using magnetic actuation to apply controlled forces on the cell membrane, in combination

with nanoparticles with different binding capabilities, biochemical pathways and ion-

channel kinetics have been studied.121

Tissue engineering and regenerative medicine are two main fields of research where the

magnetic manipulation of cells with nanomaterials is exploring new horizons. In addition

to biochemical signaling, mechanical cues provide important stimuli that promote the

production of functional tissue matrix from stem cells.122

Magnetic support-based separation and responsive hydrogel-based separation have also

been exploited in the bio-separation field, including applications as cell separation,123

enzyme immobilization,124 and protein purification125. The magnetic separation approach

has several advantages compared with conventional separation methods; it can be

performed directly in crude samples containing suspended solid materials without

pretreatment, and can easily isolate some biomolecules from aqueous systems in the

presence of magnetic gradient fields. The latter separation approach utilizes the unique

swelling properties of stimuli-responsive hydrogels to concentrate or separate dilute

biomolecule solutions.126

Recently, gene delivery and transfection were achieved both in vitro and in vivo with the

use of magnetic nanoparticles, opening the field of magnetofection: development of non-

viral transfection agents for gene delivery.127

24

1.4.6 Multifunctional nanoparticles in biomedicine

A common trend in the nanomedicine research field is to combine different

functionalities within a single nanoscale device, capable to achieve different tasks

simultaneously, such as targeting specific cells or compartments, drug delivery and

imaging.

The attachment of targeting agents to magnetic nanoparticles is used to increase the

specific accumulation of multifunctional nanoparticles within pathogenic tissues. These

structures not only deliver a drug, but also have a large surface area-to-volume ratios

allowing for a large number of therapeutic molecules to be incorporated or attached to

individual nanoparticles.15

In addition, the magnetic properties of the nanoparticles may be used for specific

targeting of these systems by external magnetic fields, to provide imaging modality for

monitoring therapeutic effects through MRI and/or to have alternative source of treatment

through magnetic fluid hyperthermia therapy.109

Multifunctional nanoparticles consisting of polymeric micelles encapsulating iron oxide

nanoparticles and fluorescent quantum dots are recently reported as MRI-fluorescent

ultrasensitive markers. 128 Mesoporous silica coated magnetic particles are also being

prepared and studied for drug delivery and simultaneous MRI imaging.129 Several other

types of multifunctional nanoparticles with the potential to integrate therapeutics and

diagnostics functions into a single nano-system have been recently developed130, both for

in-vitro studies and for in-vivo use. 131,132

Multifunctional nanoparticles are being developed more frequently for different

purposes, with the final goal of greatly improving diagnostics capabilities of several

diseases and developing specifically controlled in-situ therapy, personalized for particular

sickness and patient. The design of such multifunctional nanoparticles is the key issue in

order to achieve these goals, allowing nano-systems to be targeted to specific tissues, cells

and even intracellular compartment.133


2 Experimental

2.1 Magnetic nanoparticles

2.1.1 Synthesis of magnetic nanoparticles

In this work, two different types of iron oxide nanoparticles were studied. In the first

case, thermally blocked nanoparticles were synthesized by controlled hydrolysis of

chelate iron alkoxide complexes in alcohol solutions at elevated temperature. With this

method thermally blocked iron oxide nanoparticles, just above the superparamagnetic

limit of ca 14 nm, were prepared. A typical synthetic procedure to produce nanoparticles

with a mean diameter of 19 nm is given as an example.

Ferrous and ferric chloride were dissolved in N-methyl diethanolamine (NMDEA) and

this solution was added to a solution of sodium hydroxide dissolved in NMDEA. The

reaction mixture was mechanically stirred at room temperature for 4 h, after which the

temperature was raised slowly to 210°C and maintained for 5 h. The suspension was then

cooled to room temperature and the solid product formed was isolated by centrifugation,

washed four times with a mixture of ethanol and ethyl acetate and finally re-dispersed in

water. The synthesis was carried out in a Schlenk flask under nitrogen atmosphere.

The second type of nanoparticles prepared consists of superparamagnetic magnetite

nanoparticles (SPION) to be used in the ferrogel material. This was prepared through the

high temperature decomposition method in an organic phase and subsequently transferred

to an aqueous solution with the use of PF127.100 A typical synthetic procedure to produce

12 nm iron oxide nanocrystals via decomposition of iron oleate complex is given: solution

of ferric chloride and sodium oleate in a mixed solvent (ethanol, deionized water and

hexane) was refluxed for 4 hours. The obtained waxy Fe oleate complex was separated

and dissolved in dioctyl ether. Then the mixture, in the presence of oleic acid, was heated

to reflux for 1.5 hours, and SPION were precipitated by ethanol. Finally, the SPION were

26

dispersed in hexane in the presence of 100 μL oleic acid and stored at 4 °C for further use

and characterizations.

2.1.2 Evolution study of magnetic nanoparticles

In order to study the formation and evolution of thermally blocked nanoparticles, a

similar synthesis procedures reported above were used. Typically, 397,6 mg of

FeCl2·4H2O and 1081,2 mg of FeCl3·6H2O were dissolved in 40 g of N-methyl

diethanolamine (NMDEA). This solution was added to 40 ml 0,4 M solution of NaOH in

NMDEA in a three-neck flask under N2 atmosphere and with mechanical stirring. After 3

h, the temperature of the reaction solution was increased up to 210 °C and it was kept

constant for 5 h and 40 min, allowing the reaction to proceed. In order to study the

kinetics of the particle growth, aliquots of 1 ml of reaction mixture were taken from the

reaction vessel at different time intervals during the synthesis. These samples were taken

using a glass pipette and the reaction was quenched by cooling the reaction mixture

rapidly to room temperature.

2.1.3 Surface modification of magnetic nanoparticles

Both types of magnetic nanoparticles included in this work were surface functionalized

for the different applications. In the first case, thermally blocked nanoparticles surface

was modified with specific molecules for the analyte to be detected. As a model system

for the biomolecule detection we selected an antigen / antibodies pair. To study the

specific detection of Brucella antibodies by functionalizing the nanoparticles with

lipopolysaccarides (LPS) as corresponding antigen. Lyophilized Brucella abortus LPS

(obtained from Department for Environment, Food and Rural Affairs (DERFA), United

Kingdom) was directly immobilized on pristine nanoparticle surface. The antigen was

attached to the particles by adsorption through Van der Waals interactions, as the surface


of the particles is hydrophobic and thus there was no need for chemical coupling of

Brucella antigen.

The prepared thermally blocked nanoparticles have been exposed to high antigen

concentration (7 mg/mL), far above the critical micelle concentration (CMC) for the

antigen. A typical procedure is reported here: a small sample of suspension containing

nanoparticles (500 μL, [Fe]=4.1 mg/mL) is mixed with 100 μL of LPS solution (7 mg/mL

LPS in water) and sonicated by pulsed ultrasound cycles for 2×20 minutes.

The second type of nanoparticles, the superparamagnetic ones, has also been surface

modified to be able to transfer them in water and incorporate them in the hydrogel system.

We prepared PF127-coated water-soluble iron oxide nanoparticles by mixing a

concentrated suspension of as-synthesized SPIONs in hexane with an aqueous solution

containing an appropriate amount of PF127 followed by vigorous agitation for 30 min.

The obtained solution was dried under argon gas flow overnight, resulting in a fine

powder of POA@SPION, which is readily dispersible in water. The aqueous solution of

nanoparticles was then dialyzed against 1 L of deionized water for 48 h to remove

unbound PF127 polymers.

2.2 Ferrogel preparation

For the preparation of indomethacin (IMC) loaded ferrogel, an aqueous solution of

POA@SPION was well mixed with a PF127 solution containing an exact amount of IMC

at 4 °C to obtain a solution of PF127 (17.5%, w/v), SPION (0.5 mg Fe/mL) and IMC (1

mg/mL). The mixture was shaken to form a homogeneous suspension and the temperature

was increased to 35 °C during 10 minutes. Gelation occurred within a few minutes while

the system temperature increased. The ferrogel was cooled down again and the previous

two steps were cycled for three times. Upon gelation, the PF127 micelles and

POA@SPION organized into an ordered structure and the resulting ferrogel is formed.

28

2.3 Characterization

In order to investigate the nanoparticle morphology, size and size distribution

transmission electron microscopes were used. The analyses were performed with a JEOL

JEM-2000EX and a Philips CM200 microscope using an acceleration voltage of 200 kV.

The specimen for TEM imaging was prepared by dropping the suspension of

nanoparticles on a carbon-coated 200 mesh copper grid, followed by drying the sample

under ambient conditions. The particle size distribution of each sample was determined by

measuring the maximum dimension of at least 300 particles on TEM micrographs, and

fitting the results to probability distribution curves.

High resolution TEM analysis was also performed to study the crystal structure and

lattice orientation of the nanocrystals. In connection with TEM analysis, Link ISIS energy

dispersive x-ray (EDX) and GATAN® imaging filter (GIF) for electron energy loss

spectroscopy (EELS) and energy filtered TEM (EFTEM) were used to better understand

the chemical composition and crystal structure of the samples. Scanning electron

microscopy (SEM) was used to confirm the particle size distribution and morphology.

The specimen was prepared by centrifuging the nanoparticle suspension followed by

drying them into a vacuum oven at 70 °C overnight. SEM images were recorded at 10 kV

with a LEO 55 Ultra equipped with a field emission gun The particle size distribution was

also confirmed by photon correlation spectroscopy (PCS) measurements carried out with

a Malvern Zetasizer Nano on nanoparticle suspension.

The amount of magnetic nanoparticle in suspension was measured by taking a certain

volume of suspension and dissolving the particulate by adding hydrochloric acid to the

system. After that, the solution was diluted with water to a certain volume and the iron

concentration was then measured by atomic absorption spectroscopy (AAS - SpectrAA-

200 from Varian). The static magnetic measurements were carried out at room

temperature using a vibrating sample magnetometer (VSM-NUVO from MOLSPIN,

Newcastle Upon Tyne, UK) with a maximum applied field of 1 T.

The prepared ferrogel was also characterized by HRTEM analysis to study its structure.

The change in viscosity of the ferrogel at increasing temperature was observed using the

AR2000 rheometer (TA Instrument). For this measurement, 1.30 mL of the sample was


pipetted on the sample holder and a conical titanium plate with 40 mm in diameter was

loaded on the sample. The gap between the sample holder and the titanium plate was

maintained at 800 µm for all the measurement performed. Maintaining a constant shear

force, the sample was heated at 1 °C min-1.

2.4 Magnetic relaxation measurements

In order to estimate the relaxation of thermally blocked magnetic nanoparticles, dynamic

susceptometry was used. The measurements were performed on different nanoparticle

suspensions (as-synthesized, after functionalization with antigen and after the antigen-

antibody reaction) at room temperature. The resonance frequency for thermally blocked

particles is usually between a few Hz to few kHz while the superparamagnetic particles

relax typically via Néel process in a MHz region. For our studies, a specific AC-

susceptometer was developed by researchers at Imego AB, Göteborg. This instrument

allows measuring the susceptibility at frequencies ranging from 10 Hz to 100 kHz. The

set-up is simple since it is based on an exciting coil (in which the sample is inserted) and a

measuring one, it operates at room temperature and it requires only a few seconds to

acquire the data.

Thermally blocked nanoparticles were measured suspended in water after the synthesis

and washing procedure. Furthermore, relaxation measurements were performed on LPS

functionalized nanoparticles before and after the addition of the corresponding antibody in

solution and in serum samples. The mean hydrodynamic volume of the nanoparticles was

also calculated from the AC-susceptibility. The experimental susceptibility data were

fitted to a Debye model including the particle size distribution134 according to:

0 ( )1 ( ) H H

eff H

f D dDj D

χχωτ

=+∫

(2.1) where χ is the complex magnetic susceptibility, f(DH) the hydrodynamic size distribution

of the particles, ω the angular frequency (2πf, where f is the excitation frequency), τeff the

30

effective relaxation time of the magnetic particles (that includes both the Néel and

Brownian relaxation process) and χ0 is the DC-susceptibility.

We proposed a method to detect biomolecules directly in biological solutions based on

magnetic susceptibility measurements. The sensing principle is based on the frequency

dependence of the real and imaginary parts of the magnetic susceptibility, which is

sensitive to the increase in the hydrodynamic volume of the magnetic nanoparticles before

and after binding biomolecules. The detection scheme of the assay, depicted in Figure 2.1,

is based on a few steps: binding of specific molecules for the analyte to be detected to the

surface of the pristine thermally blocked nanoparticles, mixing the functionalized

nanoparticles to a biological sample to analyze and measuring the magnetic relaxation of

the mixture. As a result of the binding of the analyte molecules to the functionalized

nanoparticles, the hydrodynamic size is increased, which can be determined by AC

susceptometer.

AC susceptometer

Magnetic nanoparticles + LPS + serum sample

Magnetic nanoparticles added to LPS

Serum sample

Figure 2.1 Schematic illustration of the biomolecule detection scheme. First thermally blocked nanoparticles with tailored properties are synthesized, then specific molecules for the analyte to be detected are bound to the surface of the pristine nanoparticles. After this, the functionalized nanoparticles are mixed to a biological sample to analyze and finally the magnetic relaxation of the mixture is measured. A frequency shift is observed for LPS functionalized nanoparticles compared to pristine ones due to an increase in hydrodynamic volume. A further increase in volume occurs when the analyte to detect binds to the functionalized nanoparticles.

To illustrate the versatility of the method, a model system for the biomolecule detection,

Brucella abortus antigens and antibodies, was used. Aliquots of nanoparticle suspension

were mixed either with 5μl of i) positive serum (containing anti-Brucella antibodies) from

infected cows, ii) negative serum (containing no anti-Brucella antibodies) from non-

infected animal, or a mixture containing iii) 5%, iv) 10% and v) 50% positive serum. As


control sample MilliQ purified water containing NaCl, with a salt concentration

corresponding to the one in serum samples, was added to a sample with magnetic

nanoparticles. All the serum samples from animals were obtained from Department for

Environment, Food and Rural Affairs (DERFA), United Kingdom. AC-susceptibility

measurements were performed for each mixture directly after mixing.

In order to confirm the specificity of LPS-functionalized nanoparticles, further

experiments were performed with monoclonal antibodies specific to Brucella (mAb,

obtained from HyTest LtD, Finland) in buffer solution as well as with a control of non-

specific antibodies (PSA66, prostate specific antigen IgG antibodies, obtained from

CanAg Diagnostics, now Fujirebio Diagnostics, Sweden). As explained above, aliquots of

nanoparticle suspension were mixed with solution of mAb or solution of PSA66 and AC-

susceptibility was directly measured after mixing. To investigate the sensitivity of the

nanoparticle-based sensor, detection experiments were performed by diluting the original

positive serum solution 400 and 4000 times with MilliQ purified water.

2.5 Drug release rate measurements

To investigate the in vitro release of IMC drug from the ferrogel, a Franz diffusion cell

was used. The donor chamber was separated from the receptor chamber by a cellulose

membrane (MWCO: 1.2-1.4 kDa, Spectrum Inc., Breda, the Netherlands). The IMC

loaded ferrogel was pipetted into the donor chamber at 4 °C, and the temperature of the

solution was slowly increased to 37 °C. The receptor chamber was filled with 10 mL of

phosphate buffer saline (PBS, Medicago AB, Uppsala, Sweden). The release experiments

were carried out thermostatically at 37 °C throughout. A permanent magnet with field of

300 mT was used in order to examine the effect of MF on drug release rate. Two

comparative sets of experiments were undertaken; one without MF as a control and one

with the applied field. The IMC concentration was recorded by UV-Visible

spectrophotometer (Varian Cary 100 Bio, λ = 319 nm) equipped with an auto-sampler.

The data points were obtained every 5 minutes.


3 Results and discussion

3.1 Magnetic nanoparticles

3.1.1 Morphology and structure studies

Two types of magnetic nanoparticles were prepared. In the first case, thermally blocked

magnetite nanoparticles for bio-sensing application were studied. In order to investigate

the particle size, morphology and crystalline structure, TEM and SEM were used. As-

prepared iron oxide nanoparticles, i.e. pristine nanoparticles, were suspended in water and

placed on a holey carbon coated copper grid for electron microscopy analysis.

Figure 3.1 (a) shows an SEM image taken from the dried suspension of the iron oxide

nanoparticles. High resolution transmission electron microscopy (HRTEM) shows that

most of the particles are single crystals (Figure 3.1 (b)). Figure 3.2 (a) present a TEM

bright field image of a representative formulation of the pristine iron oxide nanoparticles.

5 nm

(a) (b)

Figure 3.1 (a) SEM secondary electron image of pristine iron oxide nanoparticles. Specimens were prepared by applying a few drops of the nanoparticle suspension on a standard aluminum stub. (b) High

Resolution TEM image showing a single crystal nanoparticles imaged along the zone axis [ 110 ]. Inset shows the electron diffraction pattern confirming the magnetite structure.

The average diameter of the nanoparticles was estimated in 19.5 nm (standard deviation

4 nm, as represented in Figure 3.2) as calculated measuring 300 particles and this size is

34

just above the critical superparamagnetic limit of 12-15 nm.9 The particles have a

spherical morphology, and no preferential growth direction is noticed.

5 10 15 20 25 3002468

10121416

fract

ion

of p

artic

les

[%]

median diameter [nm]

(a) (b)

Figure 3.2 (a) TEM image showing as prepared nanoparticles, (b) Size distribution analysis of the pristine nanoparticles. The mean diameter and standard deviation of the size distribution were calculated measuring 300 nanoparticles from TEM images.

The magnetite structure (Fe3O4) was confirmed by selected area electron diffraction and

energy dispersive x-ray (EDX) analysis. In addition, x-ray powder diffraction analysis

also confirmed that the nanoparticles have the inverse spinel crystal structure, typical for

magnetite and maghemite. In order to estimate the hydrodynamic size of the iron oxide

nanoparticles, Photon Correlation Spectroscopy (PCS) studies were performed and results

are shown in Figure 3.3.

0

10

20

30

0.1 1 10 100 1000 10000

Volu

me

(%)

Size (d.nm)

Size Distribution by Volume

Figure 3.3 shows PCS measurements of a representative formulation of the pristine iron oxide nanoparticles.


The results are well in agreement with the average particle size estimated from electron

microscopy analysis, confirming an average particle size of ca 20 nm with a very narrow

size distribution. The similarity between crystalline particle size (TEM analysis) and the

hydrodynamic size is consistent with the fact that no coating (i.e polymers) is present on

the nanoparticles that bear a slightly hydrophobic surface.

The second type of nanoparticles prepared is related to the drug delivery applications.

These nanoparticles, prepared by thermal decomposition of iron-oleate, have also been

characterized by TEM in order to confirm the particle size and morphology. A

representative TEM image shows that SPION in tetrahydrofuran are highly monodisperse

with as average particle diameter of 10.6 ± 0.5 nm (Figure 3.4).

Figure 3.4 TEM image of SPION prepared by thermal decomposition of iron-leate complex. A drop of particle suspension was placed and dried on a carbon-Formvar-coated 200 mesh copper grid.

SPION present a very narrow size distribution and their size is below the

superparamagnetic limit for magnetite. Most of the particles have a spherical morphology

and XRD data confirm the inverse spinel structure typical of magnetite and maghemite.

3.1.2 Evolution study

In order to better understand the formation and evolution of thermally blocked

nanoparticles, high resolution transmission electron microscopy (HRTEM) and

crystallographic analysis were performed on samples taken from the reaction mixture

36

during synthesis. Figure 3.5 shows an overview of the particles together with the

corresponding size distributions at different reaction times whereas the mean particle size

data are summarized in Table 1.

(a)

(b)

(c) Figure 3.5 TEM micrographs and the corresponding particle size distributions (particle number distribution) of magnetite nanoparticles after (a) 90 min, (b) 190 min and (c) 340 min (final sample) reaction time. The mean particle diameter is 3.6 nm in (a), 10.8 nm in (b) and 16.7 nm in (c). The particle size distribution of each sample was determined by measuring the maximum dimension of at least 300 particles from randomly chosen areas, and fitting the results to probability distribution curves


Table 1. Synthesis details and particle properties.

Reaction time Temperature (˚C) Crystalline size (nm) Best particle size distribution fit

30 min 145 3.5±0.9 lognormal

90 min 185 3.6±1 lognormal

190 min 215.6 10.8±4.2 lognormal

250 min 214.8 12.3±5.6 lognormal

310 min 215.1 12.9±3.7 normal

340 min 215.2 16.7±3.5 normal

From the data reported, it is possible to follow the formation of such nanoparticles.

Small nuclei with a mean diameter of 3.5 nm and a narrow size distribution had formed

already after 30 min and at this stage the particle size distribution could be modelled by a

lognormal distribution. No main changes occur to the nanoparticle during the next 60

minutes of reaction (Figure 3.5 a)

After 190 min reaction time, however, the maximum reaction temperature is reached

and a significant increase in particle size is recorded. The mean size is now 11 nm, and

the lognormal distribution (Figure 3.5 b) show a tail extending towards larger particle

diameters. Particles are increasing in size maintaining a spherical morphology until the

end of the reaction process and a final mean size of ca 16.7 nm with a standard deviation

of 3.5 nm (21%) is measured. A normal size distribution now gives the best fitting (Figure

3.5 c). To understand the crystalline structure of the prepared particles, lattice parameters

obtained from diffraction patterns by TEM were measured. Energy Dispersive X-ray

analysis showed a composition close to that expected for stoichiometric magnetite. The

fine structure in the electron energy loss spectra showed the characteristic peak shape of

magnetite. Further investigation based on HRTEM images of the nanoparticle formation

and crystallographic structure evolution can be found in the appended paper 2.

38

3.1.3 Magnetic study

The magnetic properties of both types of iron oxide nanoparticles prepared were studied

with different techniques. For the first type, nanoparticles for biosensor application were

magnetically characterized by static and dynamic magnetic measurements.

These analyses carried out on solutions of nanoparticles suspended in MilliQ purified

water. Static measurements performed using VSM at room temperature, are presented in

Figure 3.6.

-300

-200

-100

0

100

200

300

-0.8 -0.4 0 0.4 0.8

Mag

netiz

atio

n [A

/m]

Field B [T]

-300-200-100

0100200300

-0.10 -0.05 0.00 0.05 0.10

Figure 3.6 Magnetization versus applied field curves of pristine magnetic particle suspension in water at room temperature in two field ranges, ± 0.7 T and ± 0.1 T (the inset figure) measured using VSM.

The magnetization versus applied field curves show a sharp decrease of the

magnetization at low fields and magnetization saturates at moderate fields (about 0.4 T).

The low field magnetic susceptibility (the initial susceptibility) of nanoparticles

suspension is high compared to commercial multi core particles. This, together with the

fact that the magnetization of the nanoparticle system saturates at moderate fields, is

characteristic of the single core nature of the particles and the size of the magnetic core

(high particle magnetic moment).


To study the formation and evolution of magnetite nanoparticles, static magnetic

measurements were carried out on particles immobilized within a polymeric matrix where

the particles’ translation and rotation are inhibited. Under such conditions, the internal

single-domain relaxation, i.e. Néel relaxation, can be distinguished from Brownian

relaxation. In Figure 3.7 the magnetic measurements of particle ensembles after different

reaction times (190 min and 340 min (final sample)) are shown.

-120

-60

0

60

120

-0.15 -0.1 -0.05 0 0.05 0.1 0.15

Reaction time 190 minReaction time 340 min (final sample)

M (A

/m)

B (T)

×2

-80

-40

0

40

80

-0.01 -0.005 0 0.005 0.01

Reaction time 190 minReaction time 340 min (final sample)

M (A

/m)

B (T)

×2

Figure 3.7 Hysteresis loops in two field ranges for the particle system at two different reaction times, 190 min and 340 min, corresponding to mean particle diameters of 10.8 nm and 16.7 nm respectively. The particles are immobilized. i.e particle rotation and translation are inhibited. Only the final sample show hysteresis effects with remanence and coercivity. The magnetization values of the sample collected at 190 min reaction time are multiplied by a factor 2 for clarity.

As can be seen from Figure 3.7, the hysteresis curve for the particles formed after 190

min shows a clear superparamagnetic behaviour with no magnetic remanence and

coercivity present. As the reaction time increases (340 min) the particle size increases and

particles reach sizes larger than the superparamagnetic limit. These particles are thermally

blocked and show both magnetic remanence and coercivity in static magnetic

measurements when the particles are immobilized.

Dynamic magnetic measurements were performed using especially developed by

ImegoAC susceptometer that allows measurements of magnetic properties at much higher

frequencies than commercially available systems. It was necessary to develop a system

working over a wide frequency range because the small size of the nanoparticles used in

this study give Brownian relaxation frequencies around several tens of kHz. From the

40

measurements and the fitting procedure it is possible to calculate the hydrodynamic size

distribution of the nanoparticle system. The experimental data and the results of the fitting

are shown in Figure 3.8

0.000.010.020.030.040.050.060.070.08

10 100 1000 10000 100000

Com

plex

sus

cept

ibilit

y

Frequency [Hz]

a)

b)

Figure 3.8 AC susceptibility measurements in the range 1-250 kHz for pristine iron oxide nanoparticles. Curves a) and b) give the real and imaginary parts of the magnetic susceptibility for pristine iron oxide nanoparticles, respectively. The solid lines are obtained by fitting the data points.

Figure 3.8 shows the real (curve a) and imaginary (curve b) parts of the frequency-

dependent complex magnetization for the pristine iron oxide nanoparticles. The imaginary

part of the complex susceptibility, has a maximum at about 50 kHz for the native

nanoparticles suspended in MilliQ purified water, is due to the Brownian relaxation. From

the result of the data fitting we can determine a median diameter of the particles of about

20 nm with a size distribution width of 8 nm, which is in a good agreement with the

results from TEM data. Photon correlation spectroscopy (PCS) data also confirm the size

of the pristine nanoparticles. The magnetic, hydrodynamic and crystalline sizes are quite

similar due to the fact that the prepared nanoparticles are highly crystalline and have a

hydrophobic surface that reduces the absorption of water molecules. ‘


3.1.4 Modification of the surface of nanoparticles

For the detection of biomolecules, the thermally blocked nanoparticles were

functionalized with molecules specific for binding to the analyte to be detected. Brucella

abortus antigens and antibodies were chosen as a model compound to demonstrate the use

of thermally blocked nanoparticles for biosensor applications. The surface

functionalization of nanoparticles with Brucella abortus Lipopolysaccharide was studied

by AC-susceptibility measurements as well as by PCS.

0.000.010.020.030.040.050.060.070.08

10 100 1000 10000 100000

Com

plex

sus

cept

ibilit

y

Frequency [Hz]

a) b)c) d)

Figure 3.9 AC susceptibility measurements in the range 1-250 kHz for pristine iron oxide nanoparticles and Lipopolysaccharide (LPS) functionalized nanoparticles. Curves a) and b) give the real and imaginary parts of the magnetic susceptibility for pristine iron oxide nanoparticles, respectively. Curves c) and d) give the real and imaginary parts of the magnetic susceptibility data obtained for LPS bound-particles, respectively. A very large frequency shift is observed for LPS particles indicating a large increase in hydrodynamic volume due to LPS particles binding.

The change of AC susceptibility of the nanoparticles after LPS functionalization (Figure

3.9) shows that LPS-functionalized nanoparticles exhibit a frequency maximum at about

20 Hz, which is considerably lower than the maximum observed for pristine nanoparticles

without the LPS (about 50 kHz). This dramatic shift in frequency for the maximum in the

imaginary part of the susceptibility indicates that the hydrodynamic diameters of the LPS-

42

functionalized nanoparticles increases more than ten times as compared with that of the

pristine nanoparticles (from 20 nm to ∼260 nm).

0

10

20

0.1 1 10 100 1000 10000

Volu

me

(%)

Size (d.nm)

Size Distribution by Volume

Figure 3.10 PCS measurements of iron oxide nanoparticles suspension after functionalization with LPS. The measurements also indicate that the particle size distribution is monomodal and there is no significant amount of pristine nanoparticles in the suspension.

The hydrodynamic size of the nanoparticles determined by AC susceptometry is in

agreement with the measurements done using photon correlation spectroscopy.

For the functionalization of SPION for drug delivery applications, it was already

demonstrated that these nanoparticles are covered by a hierarchical layer composed of

PF127 and oleic acid. The PEO segments of PF127 endow SPION with hydrophilicity.100

The resulting water-soluble SPION were characterized by VSM (as shown previously in

Chapter 3.1.3)

3.1.5 Detection of biomolecules

As explained in chapter 2.4, a method for the detection of biomolecules was developed

based on the increase in hydrodynamic size of thermally blocked nanoparticles after

biding reaction of biomolecules. Brucella abortus antigens and antibodies were chosen as

model compounds. The detection method is based on the change in magnetic response of

the LPS-functionalized nanoparticles as a result of the increase of their size which is

significantly different for particles not bound to anti-Brucella antibodies (ABA). The


detailed procedure for the biomolecule sensing experiment is already reported in chapter

2.4. The magnetic susceptibility results are summarized in Figure 3.11.

0 20 40 60 80 100

260

280

300

320

340

360

380

Serum Control

Med

ian

diam

eter

[nm

]

Amount of positive serum % Figure 3.11 Hydrodynamic median diameter of LPS functionalized nanoparticles derived from magnetic susceptibility measurements versus amount of positive serum. Iron oxide nanoparticles, after LPS treatment, bind anti-Brucella antibodies contained in positive serum, resulting in an increase of their hydrodynamic volume. Repeated measurements on each samples showed virtually no spread. Error bars represent the standard deviation. The dash-line only represents the trend.

From the AC susceptibility data, there is no noticeable increase in median particle

diameter upon mixing LPS-functionalized nanoparticles suspension with negative serum.

When LPS-functionalized nanoparticles are added to positive serum, a large increase in

hydrodynamic diameter is obtained. When LPS-functionalized nanoparticles are exposed

to solutions with increasing positive serum concentrations (5%, 10% and 50%), the

hydrodynamic sizes increase accordingly.

This indicates that only antibodies specific to the LPS Brucella antigen bind to the

nanoparticles giving us the possibility to distinguish between infected and non-infected

animals. “Negative serum” refers to solutions that does not contain any Brucella

antibodies and indeed does not result in any change of nanoparticle size. However, there

might be a slight difference in total protein and antibody concentration because the serum

samples are obtained from different animals.

44

Specificity of LPS-functionalized nanoparticles was confirmed by further experiments

with monoclonal antibodies specific to Brucella as well as with a control of non-specific

antibodies (PSA66). The summary of median particle size changes obtained by AC-

susceptibility measurements are reported in Figure 3.12.

0.00 0.05 0.10 0.15 0.20

280

300

320

340

360

380

400

mAb PSA66M

edia

n di

amet

er [n

m]

mAb [mg/mL] Figure 3.12 Hydrodynamic median diameter of LPS-functionalized nanoparticles derived from magnetic susceptibility measurements. Iron oxide nanoparticles, after LPS treatment, bind anti-Brucella monoclonal antibodies mAb but do not bind non-specific PSA66 antibodies. Repeated measurements on each samples showed virtually no spread. Error bars represent the standard deviation. The dash-line only represents the trend.

The exposure of LPS-functionalized nanoparticles to the anti-Brucella antibody mAb

produces a significant shift in the maximum of the imaginary magnetic susceptibility. In

comparison, the non-specific PSA66 antibodies used as a negative control, did not give

any significant shift (see Figure 3.12). This is reflected in a large increase in

hydrodynamic size of the LPS-functionalized nanoparticles exposed to mAb antibodies as

compared with the exposure of LPS-functionalized nanoparticles to PSA66 antibodies.

The data confirm the high specificity of the LPS-functionalized nanoparticles that are

capable of sensing specifically anti-Brucella specific monoclonal antibody and not other

kind of antibodies, i.e. PSA66.

To investigate the sensitivity of the nanoparticle-based sensor, detection experiments

were performed by diluting the original positive serum solution 400 and 4000 times with

MilliQ purified water. The data didn’t show any trend in the case of 4000 times dilution.


This indicates that this nanoparticle based biosensor system is capable of detecting anti-

Brucella specific antibodies in serum with concentrations close to 0.05 μg/ml of Brucella

antibodies. The upper limit of detection (dynamic range) depends on the total number of

particles in the suspension. If we consider the lowest particle concentration that we can

detect (1 mg Fe/ml), we estimate the upper limit of detection in 0.2 mg/mL. This detection

system is therefore capable to distinguish between infected and non-infected animals

using small serum samples even at very low antibodies concentration.

3.2 Ferrogel

3.2.1 Structure study

The ferrogel system was characterized by TEM analysis and rheological measurements.

Upon gelation, the PF127 micelles organized into an ordered structure shown by TEM

(Figure 3.13). Because the specimen was not stained, only the micelles with SPION

encapsulated are visible.

The size of an individual SPION contained in the micelles is around 13 nm which is

very similar to the diameter of empty micelles formed with concentrated PF127 solutions

(> 20%). 135 The encapsulation of SPION inside the micelle seems not significantly

perturb micellar morphology or size.

Figure 3.13 TEM image of ferrogel dropped on the same type of grid at 4 °C and allowed to dry at 37 °C.

46

The TEM observation (Figure 3.13) shows that SPION loaded ferrogel constitutes of

micelles arranged in cubic liquid crystalline structures. Each micelle comprises a

hydrophobic core (PPO) in which the drug molecules are solubilized, and a hydrophilic

corona (PEO) acting as the scaffold of the ferrogel. SPION are encapsulated inside of

some micelles during the phase transfer of nanoparticles.

The rheological response as a function of temperature was examined on samples

containing 17.5% (w/v) PF127, and is reported in the text of the appended paper 3. It is

shown that, at low temperature the “sol” behaves as viscous liquid, while at high

temperature the behavior becomes elastic

The critical temperature at which the PF127 solution forms a gel depends on the

concentration of the polymer, e.g. 32.5 °C for the f containing 17.5% (w/v) PF127. The

solution remains fluidic at room temperature, while when the solution is warmed up to

human body temperature, gelation occurs and the solution becomes a solid gel. The

fluidity is thereafter restored when the gel is cooled down. In addition, the ferrogel

remains stable for few months when stored above the gelation temperature.

3.2.2 Magnetic study

The magnetic properties of both SPION and ferrogel were examined at room

temperature by using a vibrating sample magnetometer. Data are reported in Figure 3.14.

-1000 -500 0 500 1000-60

-40

-20

0

20

40

60

-100 -50 0 50 100

-30

-15

0

15

30

Mag

netiz

atio

n (A

m2 kg

-1)

Applied field (mT)

(c)

Figure 3.14 Room temperature magnetization curves of SPION (-■-) and ferrogel (-○-).


Magnetization measurements have shown that iron oxide nanoparticles retain

superparamagnetism after incorporation into the ferrogel, indicating that the magnetic

moments of particles can easily reorient upon removing the magnetic field. Saturated

magnetizations (Ms) of SPION and ferrogel are 54.1 and 39.4 Am2kg-1 respectively,

calculated considering the iron oxide content.

The lower value of Ms of the ferrogel may be attributed to the formation of the magnetic

dead layer on the surface of SPION after transfer to aqueous phase. Initial magnetic

susceptibility χ0 (measured by the slope at the M-H curve origin) for ferrogel is lower

than that of SPION, indicating less effective coupling between particles incorporated in

ferrogel caused by increased first-neighbor distance.

3.2.3 Drug release

For in vitro release test, indomethacin (IMC) has been selected as a model drug.

Practically, IMC has a very low solubility in water136, whereas in the ferrogel containing

17.5% (w/v) PF127 the concentration of IMC can be significantly increased to 1 mg/mL.

The release profiles of IMC from ferrogel are shown in Figure 3.15 both in the absence

and presence of a magnetic field.

0 2000 4000 60000

20

40

60

80

100

Cum

ulat

ive

rele

ase

(%)

Time (minute)

t1/2= 3195 min

t1/2= 1500 min

(b)

(a)

Figure 3.15 Release profiles of IMC from ferrogel in PBS (pH 7.4) at 37 °C: (a) without magnetic field; (b) with applied magnetic field.

48

For the release test without exposure to magnetic field, the drug is continuously released

up to 95% in 7000 minutes. The drug release solely depends on the diffusion of IMC

molecules from hydrophobic poly(propylene oxide) cores through water channels in the

ferrogel. The release profile is a quasi-linear curve and no significant initial burst has been

observed. As the particles are superparamagnetic, the magnetic moments of SPION are

randomly oriented within the ferrogel in the absence of magnetic field, giving a zero

magnetization throughout the gel. The release of IMC can take place through the diffusion

in the aqueous channels and the dissolution of PF127 gel.

Drug release test has also been conducted when placing a permanent magnet adjacent to

the ferrogel (5 mm away from the gel, magnetic field is ca. 300 mT). The concentration of

released IMC increases much faster in the presence of external magnetic field. The release

half-time (t1/2) is reduced to 1500 minutes from 3195 minutes when measured without

applied field.


4 Conclusions

In this work, we developed tailored nanoscale magnetic nanoparticles for advanced bio-

applications, such as the detection of biomolecules directly in biological samples and a

novel injectable drug delivery system.

For a sensitive and selective detection of biomolecules, magnetite nanoparticles with

specific magnetic properties were developed and used in a novel detection scheme that is

fast, easy to use and sensitive. Thermally blocked magnetic nanoparticles are the key

component in this detection method. The prepared nanoparticles showed excellent

magnetic properties for this application, as well as a narrow size distribution and good

crystallinity. The sensing system was able to detect the changes of the magnetic properties

of the nanoparticles as indication of the presence of functional molecules on its surface.

Such functionalized system was capable of sensing biomolecules, such as Brucella

antibodies, directly in biological samples, which can be used for distinguishing between

infected and non-infected animals. In-depth studies of the formation of such nanoparticles

were performed by correlating electron microscopy analysis with magnetic data at

different stages during the synthesis procedure. The developed thermally blocked

magnetic nanoparticles are the key point of the detection system, providing very good

sensitivity and a versatile platform for bio-conjugation and detection of different

biomolecules.

Beside the work on diagnostic tools, a novel drug delivery system based on magnetically

enhanced release of drug was prepared and studied. The system is based on the integration

of superparamagnetic iron oxide nanoparticles and Pluronic F127 gels. Indomethacin, a

hydrophobic drug, was loaded in the ferrogel and the characteristic sol to gel

transformation property was studied. Drug release tests showed that it is possible to

enhance the release rate by applying an external magnetic field. This, together with the

fact that the sol to gel transformation temperature can be tailored to be at body

temperature (36 °C), makes it a very interesting material as injectable drug delivery

system, compared to traditional gels that have to be surgically implanted.

50

Future work

In the future, more experiments related to other biomolecules detection will be

undertaken, as well as other magnetic detection systems will be tested with the developed

thermally blocked nanoparticles. Further work will be focused on loading the ferrogel

with other drugs and with other types of magnetic and fluorescent nanoparticles. In vitro

and possibly in vivo studies of the cytotoxicity and body response will be performed with

the developed ferrogel.

Besides this, a lot of effort will be focus on developing other magnetic nanostructures,

such as different nanoparticles and single-element or multi-segment nanowires, for

various applications.

The synthesis of multifunctional nanoparticles will also be undertaken, aiming at the

preparation of polymeric nanostructures that will carry therapeutic compounds, magnetic

nanoparticles for MRI visualization, fluorescent probes for microscopy studies and

targeting molecules to achieve specific uptake.


Acknowledgements

First and foremost, I would like to thank my supervisor, Prof. Mamoun Muhammed,

for introducing me to the world of scientific research, and in particular to the field of

nanotechnology and materials science. I have also to thank him for the opportunity he

gave me to join the Functional Materials Division. His ideas and kindness are always a

strong support during my studies.

I want to thank Dr. Muhammet Toprak for his great help from research to everyday life,

especially for his kind help in revising this thesis. I wish him a great success in the

scientific career. Thousands of thanks to Jian Qin, Sylvan, for his tremendous help in

planning experiments, lab work and fruitful discussions. I wish him all the best for his

PhD and his future as bright scientist and as great dad! Thousands of thanks also to Dr.

Shanghua Li, Peter, for his help, discussion and support during this time. I wish him as

well all the best for his future career, together with his family. Many thanks to Carmen,

Sverker, Fei, Abhi, Stefano, Salam and all other members in Functional Materials

Division for their priceless friendship. I wish all the best to all of them, both for their

research and for their life. I would like to acknowledge the fruitful collaboration and

discussions with Karolina Petersson, Dr. Anatol Krozer and Dr. Christer Johansson from

Imego research institute in Göteborg, and Stefan Gustafsson and Prof. Eva Olsson from

the Department of Applied Physics, Chalmers University of Technology.

Special thanks to all the friends, Italians and non-Italians, which I met here in

Stockholm for the unforgettable time we had and still have together. It will be too long to

mention all of them.

I would like to deeply thank my parents, my brother and my grandma for their endless

support. Grazie e mille per tutto quello che avete fatto e continuate a fare per me.

Finally, I have to thank the most important person in my life, my wonderful fiancée

Olesja, for her endless love, support and strength, even in difficult times. Without her all

this would not be possible and my life would not be the same. Thanks for brightening up

my days and being always next to me.


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