Synthesis of Nanostructured and Hierarchical Materials for Bio-Applications Fei Ye 叶飞 Licentiate Thesis Stockholm 2011 Division of Functional Materials School of Information and Communication Technology Royal Institute of Technology
Synthesis of Nanostructured and Hierarchical Materials for
Bio-Applications
Fei Ye
叶飞
Licentiate Thesis
Stockholm 2011
Division of Functional Materials
School of Information and Communication Technology
Royal Institute of Technology
Postal address Division of Functional Materials
School of Information and
Communication Technology
Royal Institute of Technology
Electrum 229
Isafjordsgatan 22
SE 164 40, Kista, Sweden
Supervisor Prof. Mamoun Muhammed
Email: [email protected]
TRITA-ICT/MAP AVH Report 2011:04
ISSN 1653-7610
ISRN KTH/ICT-MAP/AVH-2011:04-SE
ISBN 978-91-7415-903-5
© Fei Ye, 2011
Kista Snabbtryck AB, Kista 2011
Functional Materials Division, KTH, 2011 i
Abstract In recent years, nanostructured materials incorporated with inorganic particles
and polymers have attracted attention for simultaneous multifunctional biomedical
applications. This thesis summarized three works, which are preparation of
mesoporous silica coated superparamagnetic iron oxide (Fe3O4@mSiO2)
nanoparticles (NPs) as magnetic resonance imaging T2 contrast agents, polymer
grafted Fe3O4@mSiO2 NPs response to temperature change, synthesis and
biocompatibility evaluation of high aspect ratio (AR) gold nanorods.
Monodisperse Fe3O4@mSiO2 NPs have been prepared through a sol-gel process.
The coating thickness and particle sizes can be precisely controlled by varying the
synthesis parameters. Impact of surface coatings on magnetometric and relaxometric
properties of Fe3O4 NPs is studied. The efficiency of these contrast agents, evaluated
by MR relaxivities ratio (r2/r1), is much higher than that of the commercial ones. This
coating-thickness dependent relaxation behavior is explained due to the effects of
mSiO2 coatings on water exclusion.
Multifunctional core-shell composite NPs have been developed by growing
thermo-sensitive poly(N-isopropylacrylamide-co-acrylamide) (P(NIPAAm-co-AAm))
on Fe3O4@mSiO2 NPs through free radical polymerization. Their phase transition
behavior is studied, and their lower critical solution temperature (LCST) can be subtly
tuned from ca. 34 to ca. 42 ˚C, suitable for further in vivo applications.
A seedless surfactant-mediated protocol has been applied for synthesis of high
AR gold nanorods with the additive of HNO3. A growth mechanism based on the
effect of nitrate ions on surfactant micelle elongation and Ostwald ripening process is
proposed. The biocompatibility of high AR nanorods was evaluated on primary
human monocyte derived dendritic cells (MDDCs). Their minor effects on viability
and immune regulatory markers support further development for medical applications.
Keywords: Fe3O4, mSiO2, core-shell, MRI, multifunctional, PNIPAAm, LCST, gold,
nanorod, nitric acid, AR, MDDC, biocompatibility, immunomodulation
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Functional Materials Division, KTH, 2011 iii
LIST OF PAPERS
This thesis is based on following publications: 1. Fei Ye, Helen Vallhov, Jian Qin, Evangelia Daskalaki, Abhilash Sugunan,
Muhammet S. Toprak, Andrea Fornara, Susanne Gabrielsson, Annika Scheynius and Mamoun Muhammed, “Synthesis of high aspect ratio gold nanorods and their effects on human antigen presenting dendritic cells”, in press, International Journal of Nanotechnology, 2011
2. Fei Ye, Jian Qin, Muhammet S. Toprak and Mamoun Muhammed,
“Multifunctional core-shell nanoparticles: superparamagnetic, mesoporous, and thermo-sensitive”, in press, Journal of Nanoparticle Research, 2011, DOI: 10.1007/s11051-011-0272-8
3. Andrea Kunzmann, Britta Andersson, Carmen Vogt, Neus Feliu, Fei Ye, Susanne
Gabrielsson, Muhammet S. Toprak, Tina Thurnherr, Sophie Laurent, Marie Vahter, Harald Krug, Mamoun Muhammed, Annika Scheynius and Bengt Fadeel, “Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells”, in press, Toxicology and Applied Pharmacology, 2011, DOI: 10.1016/j.taap.2011.03.011
Other work not included: 1. Andrea Fornara, Alberto Recalenda, Jian Qin, Abhilash Sugunan, Fei Ye, Sophie
Laurent, Robert N. Muller, Jing Zou, Abo-Ramadan Usama, Muhammet S. Toprak and Mamoun Muhammed, “Polymeric/inorganic multifunctional nanoparticles for simultaneous drug delivery and visualization”, Materials Research Society Symposium Proceedings Vol. 1257, 2010, 1257-O04-03
2. Lin Dong, Fei Ye, Jun Hu, Sergei Popov, Ari T. Friberg and Mamoun Muhammed,
“Fluorescence quenching and photobleaching in Au/Rh6G nanoassemblies: impact of competition between radiative and non-radiative decay”, Journal of the European Optical Society − Rapid Publications, 2011, 6, 11019
3. Fei Ye, Sophie Laurent, Jian Qin, Alain Roch, Muhammet S. Toprak, Robert N.
Muller and Mamoun Muhammed, “Uniform mesoporous silica coated iron oxide nanoparticles as highly efficient MRI T2 contrast agents with tunable proton relaxivities”, Manuscript
iv
Conference presentations 1. Fei Ye, Jian Qin, Muhammet S. Toprak and Mamoun Muhammed,
“Multifunctional core-shell nanoparticles: superparamagnetic, mesoporous, and thermo-sensitive”, 10th International Conference on Nanostructured Materials, Sept 13-17, 2010, Rome, ITALY (poster)
2. Daniel Bacinello, Andrea Fornara, Jian Qin, Fei Ye, Muhammet Toprak and
Mamoun Muhammed, “Laser triggered drug release from smart polymeric nanospheres containing gold nanorods”, 10th International Conference on Nanostructured Materials, Sept 13-17, 2010, Rome, ITALY (poster)
3. Fei Ye, Jian Qin, Sophie Laurent, Muhammet S. Toprak, Robert N. Muller and
Mamoun Muhammed, “Uniform mesoporous silica coated superparamagnetic iron oxide nanoparticles for T2-weighted magnetic resonance imaging”, 12th Bi-Annual Conference on Contrast Agents and Multimodal Molecular Imaging, May 19-21, 2010, Mons, BELGIUM (poster)
4. Andrea Fornara, Alberto Recalenda, Jian Qin, Abhilash Sugunan, Fei Ye, Sophie
Laurent, Robert N. Muller, Jing Zou, Abo-Ramadan Usama, Muhammet S. Toprak and Mamoun Muhammed, “Polymeric/inorganic multifunctional nanoparticles for simultaneous drug delivery and visualization”, Materials Research Society Spring meeting (2010 MRS), Apr 5-9, 2010, San Francisco/California, USA
5. Carmen Vogt, Andrea Kunzmann, Britta Andersson, Fei Ye, Neus Feliu Torres,
Tina Thurnherr, Sophie Laurent, Jean-Luc Bridot, Robert Muller, Muhammet Toprak, Harald F. Krug, Annika Scheynius, Bengt Fadeel and Mamoun Muhammed, “Tunable superparamagnetic Fe3O4-SiO2 core-shell nanoparticles: synthesis, characterization, and in vitro compatibility with immune-competent cells”, 34th International Conference and Exposition on Advanced Ceramics and Composites, Jan 24-29, 2010, Daytona Beach, Florida, USA
6. Lin Dong, Jun Hu, Fei Ye, Sergei Popov, Ari T. Friberg, Mamoun Muhammed,
“Influence of nanoparticles concentration on fluorescence quenching in gold/Rhodamine 6G nanoassemblies”, 2009 Asia Communications and Photonics Conference and Exhibition, Nov 2-6, 2009, Shanghai. CHINA
7. Fei Ye, Abhilash Sugunan, Jian Qin and Mamoun Muhammed, “A general method
for preparation of mesoporous silica coating on gold nanorods, nanoparticles, and quantum dots”, EuroNanoMedicine 2009, Sept 28-30, 2009, Bled, SLOVENIA (poster)
8. Stefan Gustafsson, Andrea Fornara, Fei Ye, Karolina Petersson, Christer
Johansson, Mamoun Muhammed and Eva Olsson, “TEM investigation of magnetic nanoparticles for biomedical applications”, in Materials Science, edited by Silvia Richter and Alexander Schwedt (14th European Microscopy Congress,
Functional Materials Division, KTH, 2011 v
Aachen, Germany, 2008) Vol. 2: Materials Science, pp. 209-210, DOI: 10.1007/978-3-540-85226-1_105
9. Fei Ye, Jian Qin and Mamoun Muhammed, “Cellular uptake and cytotoxicity of
gold nanorods”, Nanoimmune Workshop, Feb 13, 2007, Uppsala, SWEDEN
vi
Contributions of the author
Paper 1. Planning of experiments, preparing the materials, performing material characterization, evaluation of the results, and writing main part of the article. Paper 2. Planning of experiment, preparing the materials, performing material characterization, evaluation of the results, and writing the article. Paper 3. Preparing the materials, performing material characterization, and writing parts of the article.
Functional Materials Division, KTH, 2011 vii
ABBREVIATIONS AND SYMBOLS
AAm acrylamide AAS atomic absorption spectroscopy AR aspect ratio ATRP atom transfer radical polymerization BET Brunauer-Emmett-Teller CT computed X-ray tomography CTAB cetyltrimethylammonium bromide DSC differential scanning calorimetry EtOAc ethyl acetate FDA Food and Drug Administration HRTEM high resolution transmission electron microscopy ICP-AES
inductively coupled plasma-atomic emission spectrometry
IR infrared LCST lower critical solution temperature [˚C] LPS lipopolysaccharide M magnetization [emu g-1] MBA N,N’-methylene bisacrylamide MCM Mobil crystalline materials MDDC monocyte-derived dendritic cells MR magnetic resonance MRI magnetic resonance imaging NIPAAm N-isopropylacrylamide NIR near infrared NLDFT nonlocal density functional theory NMR nuclear magnetic resonance NMRD nuclear magnetic resonance dispersion NP nanoparticle PCS photon correlation spectroscopy PDLA poly(D,D-lactide) PEG poly(ethylene glycol) PET positron emission tomography PLGA poly(lactic acid-co-glycolic acid) PLLA poly(L,L-lactide) PNIPAAm poly(N-isopropylacrylamide) P(NIPAAm-co-AAm) poly(N-isopropylacrylamide-co-acrylamide) QD quantum dot RAFT reversible addition fragmentation chain transfer RES reticuloendothelial system RF radio frequency
viii
SAED selected area electron diffraction SPECT single-photon-emission computed tomography SPIO superparamagnetic iron oxide SPION superparamagnetic iron oxide nanoparticle SPR surface plasmon resonance TEM transmission electron microscopy TEOS tetraethyl orthosilicate TMSMA 3-(trimethoxylsilyl) propyl methacrylate TSC trisodium citrate VSM vibrating sample magnetometer XRD X-ray diffraction σ standard deviation
τD translational correlation time [s]
τN Néel relaxation time [s]
Functional Materials Division, KTH, 2011 ix
Table of contents
ABSTRACT…………………………………………………………………………...I
LIST OF PAPERS…………………………………………………………………..III
ABBREVIATIONS AND SYMBOLS…………………………………………….VII TABLE OF CONTENTS…………………………………………………………...IX 1 INTRODUCTION.................................................................................................1
1.1 OBJECTIVES ..............................................................................................................1 1.2 OUTLINE ....................................................................................................................2 1.3 MULTIFUNCTIONAL NANOSTRUCTURES ..........................................................3
1.3.1 Core-shell or micellar structure ............................................................................4 1.3.2 Porous structure ....................................................................................................5 1.3.3 Heterodimer or core-satellite structure.................................................................6
1.4 FUNCTIONAL NANOMATERIALS FOR BIOMEDICAL APPLICATIONS ..........7 1.4.1 Drug carriers ........................................................................................................7 1.4.2 Imaging probes......................................................................................................8 1.4.3 Thermo-therapy agents .........................................................................................9 1.4.4 Stimuli-sensitive drug delivery agents.................................................................10
1.5 BIOMEDICAL APPLICATIONS OF NANOMATERIALS .....................................11 1.5.1 Magnetic resonance imaging ..............................................................................11
1.5.1.1 Physical background ................................................................................11 1.5.1.2 Contrast agents .........................................................................................12 1.5.1.3 Superparamagnetism ................................................................................13 1.5.1.4 Preparation methods for magnetic NPs ....................................................13
1.5.2 Plasmonic photothermal therapy ........................................................................15 1.5.2.1 Surface plasmon resonance ......................................................................15 1.5.2.2 Preparation methods for gold NPs and NRs.............................................15
1.5.3 Temperature-triggered drug release....................................................................16 1.5.3.1 Thermo-responsive phase transition.........................................................16 1.5.3.2 Preparation methods for thermo-sensitive polymers ................................17
2 EXPERIMENTAL ..............................................................................................18 2.1 MAGNETIC MESOPOROUS NANOPARTICLES..................................................18
2.1.1 Synthesis of SPION and phase transfer...............................................................18 2.1.2 Fabrication of Fe3O4@mSiO2 .............................................................................18
2.2 PREPARATION OF THERMO-SENSITIVE COMPOSITE NANOPARTICLES...19 2.3 SYNTHESIS OF GOLD NANORODS.....................................................................19 2.4 CHARACTERIZATIONS..........................................................................................20 2.5 BIOCOMPATIBILITY EVALUATION.....................................................................21
2.5.1 Co-culture immature MDDCs with gold nanorods and spheres .........................21 2.5.2 Flow cytometry analysis......................................................................................21 2.5.3 Cell viability assay ..............................................................................................21
3 RESULTS AND DISSCUSSIONS .....................................................................22 3.1 SUPERPARAMAGNETIC MESOPOROUS CORE-SHELL NANOPARTICLES ..22
3.1.1 Morphology study ...............................................................................................22 3.1.2 Characterization of mesoporous silica................................................................24 3.1.3 Magnetometric and relaxometric properties .......................................................25 3.1.4 MR studies of Fe3O4@mSiO2 nanoparticles .......................................................26
3.2 MULTIFUNCTIONAL CORE-SHELL COMPOSITE NANOPARTICLES ............28 3.2.1 Morphological and structural studies .................................................................28
x
3.2.2 Magnetic properties ............................................................................................30 3.2.3 Thermo-responsive properties and manipulation of LCST .................................31
3.3 HIGH ASPECT RATIO GOLD NANORODS ..........................................................34 3.3.1 Morphological and structural studies .................................................................34 3.3.2 Particle growth mechanism studies.....................................................................35 3.3.3 Biocompatibility evaluation of high aspect ratio gold nanorods ........................38
3.3.3.1 Viability studies........................................................................................38 3.3.3.2 Immune modulatory effects......................................................................39 3.3.3.3 Cellular internalization study ...................................................................41
4 CONCLUSIONS .................................................................................................42
FUTURE WORK.......................................................................................................43
ACKNOWLEDGEMENTS ......................................................................................44
REFERENCES...........................................................................................................45
Functional Materials Division, KTH, 2011 1
1 Introduction
Nanotechnology, a concept first introduced by Richard Feynman during his talk
in December 1959,1 has implications from the medical, ethical, and environmental
applications, to many fields such as materials science, engineering, biology, chemistry,
physics, computing, and communications. Nanomaterials, feature in nanoscale size,
are key components for the development of nanotechnology due to the extraordinary
physical and chemical properties stemming from their nanoscale dimensions. Owing
to the similar size to that of most biological molecules and structures, nanomaterials
have been exploited by the biological and medical research communities for their
unique properties for various applications. Along with the tremendous development of
this research field, terms such as biomedical nanotechnology, nanobiotechnology, and
nanomedicine are coined to describe hybrid of the fields.2 Thus far, the integration of
nanomaterials with biology has led to numerous developments, such as diagnostic
devices, contrast agents, analytical tools, physical therapy applications, and drug
delivery vehicles, etc.3
1.1 Objectives
In this thesis, we are aiming to develop multifunctional NPs used in biomedical
applications, primarily as a contrast agent for MRI, temperature-responsive
biomaterials for magnetic separation and drug release, as well as evaluating the
biocompatibility of the nanomaterials.
1). SPION was synthesized in non-polar solvent and in order to use it as MRI
contrast agents, CTAB is employed as a phase transfer agent to render the dispersity
of SPIONs in aqueous phase. Mesoporous silica (mSiO2) coating layers have been
grown on surface of SPIONs via the organic template of CTAB. The dependence of
magnetic relaxivities and contrast enhancing properties of the core-shell NPs on the
thickness of the SiO2 coating layer are evaluated.
2). To incorporate thermosensitive property with core-shell magnetic
2
mesoporous NPs, for temperature-triggered magnetic separation or potential
temperature-controlled drug release applications, PNIPAAm is used for response to
temperature variation and a co-monomer of AAm for tuning this property.
3). As another type of nanomaterials for biomedical applications, gold nanorods
with high AR are prepared through a non-seeded and surfactant-mediated method.
Prior to further medical applications, the biocompatibility of the high AR gold
nanorods is evaluated by the viability and immune modulatory analyses on human
primary MDDCs.
1.2 Outline
This thesis deals with the development of multifunctional NPs for various
biomedical applications, such as MRI and magnetic separation, and potential
temperature-sensitive drug release, and thermal therapy.
Chapter 1.3 briefly introduces several frameworks for the construction of
hierarchical nanomaterials with simultaneous multifunctions, e.g. visualization,
environment-sensitivity, loading, targeting, and photothermal heating. Chapter 1.4
gives an overview on different categories of nanomaterials as drug carriers, e.g.
mesoporous materials, polymer particles, or liposomes; imaging and thermo-therapy
agents, e.g. iron oxide, or gold; and temperature-sensitive moieties, e.g. PNIPAAm.
Chapter 1.5 briefly presents the physical background of some biomedical
applications and the use of the functional nanomaterials.
Chapter 2.1 starts with the preparation methodology of SPIONs and mSiO2
coated Fe3O4 (Fe3O4@mSiO2) core-shell NPs. In Chapter 2.2, synthesis and grafting
methods of thermo-sensitive polymers are summarized. Synthesis of gold nanorods is
described in Chapter 2.3. Following characterization of physico-chemical properties
of materials by different techniques in Chapter 2.4, biocompatibility evaluation is
reported in Chapter 2.5.
Chapter 3 discusses the results of synthesis and characterization of nanomaterials.
In Chapter 3.1, the morphology and structure of Fe3O4@mSiO2 core-shell NPs with
Functional Materials Division, KTH, 2011 3
variable coating thicknesses are discussed. The efficiency of these NPs as MRI T2
contrast agents are evaluated on the magnetic relaxivities and the impacts of mSiO2
coating thickness are also studied. In Chapter 3.2, thermo-sensitive polymer
PNIPAAm coated Fe3O4@mSiO2 NPs (designated as Fe3O4@mSiO2@PNIPAAm) are
studied with respect to magnetism, porosity, and thermal behaviors. LCST of
Fe3O4@mSiO2@PNIPAAm NPs can be manipulated by adjusting the composition of
the monomers for polymerization. Chapter 3.3 discusses the effect of nitric acid on
the crystal structures of the initially formed nuclei and consequently the growth of
gold nanorods. The biocompatibility of high AR gold nanorods was evaluated by
testing their viability and immune modulatory effects on human primary MDDCs,
which were compared with the effects caused by low AR gold nanorods.
1.3 Multifunctional nanostructures
Multifunctional nanomaterials, incorporating several components with different
functions into one entity, have attracted plenty of interest due to their combined
functionalities capable for simultaneously achieving multiple tasks, which can be
advantageously used as compared to components with a single function. Numerous
studies have been conducted on the development of multifunctional NPs as tools for
imaging,4-10 drug delivery,9-12 biosensing,13, 14 cancer therapy,5-8 and magnetic cell
separation.15 Obviously, a smart combination of the principles would lead to novel
tools with many applications in life sciences.
In some applications, appropriate designs of the nanostructures are required to
achieve specific functionalities. For example, multifunctional nanomaterials capable
to respond to external stimuli, such as changes of temperature,16 pH value,17
photoirradiation,18 ultrasound,19 biomolecules,20 and magnetic field,21 need sensitive
moieties exposed to or permeable to the external sources of excitation. In MRI
applications, in order to enhance T1 signals, paramagnetic electron spins of the T1
contrast agent are required to be directly in contact with water molecules via dipolar
interactions between electron spins of the contrast agent and nuclear spins of water.22,
4
23 PEG modification of NPs without affecting the characteristics of the NPs is also
necessary to avoid recognition by the RES prior to reaching targeting tissues.24
Biocompatibility is another key factor for the implants in medical applications, which
means good blood compatibility and low toxicity.25
1.3.1 Core-shell or micellar structure
Core-shell structure is one of the most common types of assembly of NPs
bringing together different functional materials into one entity (Figure 1.1a). The core
normally is a solid NP of various materials such as metal,26-28 oxides,9, 29-31 or
polymers.32-34 In most of the cases, the functionalities of the core NPs are retained in
the presence of coating layers. In some other applications, the core particles act as a
sacrificial template for grafting or growing the shell materials with certain
morphology or functionality, and then are removed by dissolution,35 etching,36-38 or
decomposition through calcination39/UV irradiation40 in order to form cavities to load
drugs in the interior space. Alternatively, the sandwiched layer as the barrier of cores
also can be removed to facilitate the retention of physical properties of core
materials41 or the transmission of external stimuli to the core.42 Instead of solid cores,
the microemulsion method has been used to prepare nanostructures with soft cores of
oil beads or hydrophobic domain of polymers,43, 44 which contain hydrophobic interior
NPs or can be encapsulated with hydrophobic drugs. Liposomes45, 46 (Figure 1.1b) and
Figure 1.1 Schematic representations of multifunctional NPs with (a) a core-shell structure47 combined with targeting, imaging, cell-penetrating, stimulus-selective agents, therapeutic compounds, and a stabilizing polymer to ensure biocompatibility; (b) a liposome,45 and (c) a typical spherical micelle48 structure.
Functional Materials Division, KTH, 2011 5
micelles48-51 (Figure 1.1c) with core-shell structures are also functional materials used
for therapeutic applications. The unique structure of liposome with hydrophilic
interior and hydrophobic lipid double layer facilitates the incorporation of hydrophilic
and hydrophobic drugs, respectively. They are protected from degradation and safely
transported until the liposome reaches the infected area, where the temperature is
higher than the body temperature and the structure is destabilized to release the
payloads.52 Micelles or reverse micelles are composed of monolayers of surfactant
molecules53 or amphiphilic polymers.48 Besides the manipulation of their
morphologies,54, 55 micelles with multifunctional modalities have been developed for a
variety of nanomedicine applications.56
1.3.2 Porous structure
Porous structure allows the integration of different materials in the pores, and it is
another type of system for simultaneous realization of multiple tasks. To arrange
multi-components in porous structures, NPs can be coated by porous materials (Figure
1.2a), or distributed inside the pores (Figure 1.2b). In the first construction, porous
layers are grown in situ around the NP cores along their surface-capping agents,
which may act simultaneously as organic templates for porous structures.9, 57 Or the
porous structure is formed by subsequent etching of the condensed shell materials.58
In another way, porous materials are template-synthesized utilizing surfactants,59
block copolymers,60, 61 or salts62 as sacrificial pore templates, and other materials can
be grown or loaded in the pores. Other methods, including replication of porous
alumina,63 liquid-crystal templates,64 or dealloying,65 to fabricate nanoporous metals
Figure 1.2 Schematic diagrams of porous multifunctional structures with (a) NPs as coated cores,9 or (b) NPs distributed in cavities of porous materials.66, 67
6
are reported. Very recently, non-toxic porous metal–organic frameworks have been
developed for biomedical applications.68
1.3.3 Heterodimer or core-satellite structure
Heterodimers are dumbbell-like NPs with two different functional NPs in
intimate contact (Figure 1.3a).69 They are commonly synthesized by sequential
growth of a second component on premade seeds through anisotropical nucleation
centered on one specific crystal plane of the seeding particles and their well-matched
lattice spacing lower the energy for epitaxial nucleation of second component. The
critical condition for growing heterodimmer NPs relies on promoting heterogeneous
nucleation of a second material while suppressing its homogeneous nucleation,70, 71
which results in the formation of separated NPs or core-shell structures. Other
methods to assemble different components together, instead of direct matching the
crystal planes in dimer NPs, include immobilizing or coordinating a second
component with the substrate particles through chemical linkage to form core-satellite
morphology (Figure 1.3b).72-74 The incorporation of different functional materials
with controlled surface modifications into one unit is a promising approach for
designing multifunctional diagnostic and therapeutic applications.
Figure 1.3 Schematic illustrations of multifunctional (a) heterodimer NPs69 and (b)
NPs with core-satellite structures.72
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1.4 Functional nanomaterials for biomedical applications
1.4.1 Drug carriers
Actively targeted drug carriers with entrapped drugs are capable of increasing the
therapeutic efficacy of a drug and reducing its systemic side-effects by delivering the
drug to the diseased site.75 Surface modification, e.g. folate modification, of the drug
carriers is necessary for targeting drug delivery.76, 77 Moreover, an ideal drug carrier
should not induce any immune response and should be degradable and produce
nontoxic degradation products. Hydrodynamic size of drug carriers is related with
their capabilities on overcoming the biological defense system and vascular barriers
for therapeutic delivery.78, 79 Furthermore, PEGylation of the drug carriers inhibits the
uptake by the RES and the blood circulation time increases.80
Drug carriers can be divided into two broad classes, depending on the interactions
between the drug and carrier, as macromolecular conjugates and particulate drug
carriers.81 In macromolecular conjugations, the drugs including proteins, antibodies,
and oligonucleotides are chemically linked to synthetic, natural, or pseudosynthetic
polymers. The chemical linker between the drug and the carrier is normally
acid-cleavable or reduction-sensitive, which will influence the controlled release of
the drug.82 Another category of drug carriers is particulate carriers that entrap the drug
in a loading space, thus isolating the drug from the environment and providing a high
degree of protection from enzymatic inactivation. Since covalent conjugation is not
necessary for entrapping the drug,83 a single carrier can be loaded with various drugs.
The most intensively investigated particulate drug carriers are liposomes and
polymeric NPs. Some of the medical applications of liposomes have reached the
preclinical stage and a few of liposomal formulations have been approved by FDA.84,
85 Among polymeric particles, biocompatible and biodegradable polyesters, such as
PLLA, PDLA, and PLGA, are widely studied.86 Hydrogels, another potential drug
carrier, can protect the drug from hostile environments and also control drug release
by changing the gel structures in response to environmental stimuli.87 Mesoporous
8
materials are also used as drug carriers with the features of high surface area/pore
volume and ordered pore network. Specifically, for mesoporous silica particles, their
silanol-containing surface can be functionalized for control of drug loading and
release.88-90
1.4.2 Imaging probes
Molecular imaging, with analytic and diagnostic ability at the molecular or
cellular level, has emerged recently which combines the molecular biology and in
vivo imaging.91, 92 Representative non-invasive imaging techniques include optical
imaging, CT, MRI, PET, SPECT, and ultrasound,93 which allow real-time
visualization of cellular functions of living organisms. Efforts have been devoted to
develop the imaging probe or contrast agent that can improve the sensitivity and
detectability of the current imaging tools for diagnosing disease,94 monitoring disease
progression,95 and tracking therapeutic response.96 Besides the available imaging
probes and contrast agents of organic dye,9 chelate,97 or radioactive agents,96 new
types of probes based on inorganic NPs have been developed, because of their useful
optical and magnetic properties derived from their compositions and nanometer
sizes.98 For instance, QDs show advantages in biological imaging, such as
size-tunable absorption and emission, flexibility in excitation due to broad and intense
absorption, and high fluorescence quantum yields even in the NIR wavelengths as
compared to organic dyes.99 SPION100, 101 are clinically available, relatively benign
contrast agents for MRI with good sensitivity and low toxicity. Metal-doped ferrite
NPs, e.g. superparamagnetic manganese ferrite (MnFe2O4),102 can induce significant
MR contrast-enhancement effects, especially useful for MRI of small cancers.103 Iron
oxides can also be modified through radiolabeling104, 105 for PET or SPECT as a
multimodal imaging probe. Some other magnetic NPs, such as metal alloys of FeCo
and FePt, Gd2O3,106 and MnO,107 are successfully utilized for in vitro cell labeling and
in vivo T1-weighted MRI.108, 109 Metallic gold nanostructures, generally NP, nanorod,
nanoshell, and nanocage, are efficient contrast agents in optical imaging based on
Functional Materials Division, KTH, 2011 9
their unique SPR properties derived from the interaction of electromagnetic waves
with the electrons in the conduction band.110 This SPR enhances all linear and
nonlinear optical properties and thus offers multiple imaging modalities including
light scattering,111 extinction,112 two-photon luminescence,113 multiphoton
imaging,114 photothermal,115 and photoacoustic imaging.116 Efforts have been devoted
to the tuning of SPR imaging properties of Au NPs by morphological or surface
modifications117 and investigation of their surface interactions with cells.118, 119
1.4.3 Thermo-therapy agents
Magnetic NPs, mainly biocompatible SPION, have shown applications for cancer
treatment utilizing the locally generated heat, i.e. hyperthermia, to induce necrosis of
tumor cells using the energy absorbed from an oscillating magnetic field.120, 121
Clinical trials on feasibility of magnetic hyperthermia have been conducted for
patients with prostate cancers and a significant inhibition of prostate cancer growth
was found.122, 123 Other magnetic metallic NPs of Co, Fe or FeCo, with higher
magnetization compared to iron oxide, have been investigated for their hyperthermia
properties.124, 125
Another type of tumor treatment is by photodynamic therapy (PDT), which
involves cell destruction caused by toxic singlet oxygen or other free radicals that is
initiated by the reaction of a photosensitizer with tissue oxygen upon exposure to a
specific wavelength of light in the visible or NIR region.126 However, a major
drawback of PDT is that the photosensitizing drug stays in the body for a long time,
rendering the patient to be highly sensitive to light. An alternative to PDT is the
photothermal therapy (PTT) in which photothermal agents are employed to achieve
the selective heating of the local environment. Noble metal NPs, including gold
nanospheres,127 nanorods,111 nanoshells,128 and nanocages,129 are superior than
conventional photoabsorbing dyes as agents for PTT on account of their enhanced
absorption cross sections, which implies effective laser therapy at relatively lower
energies for minimal invasion, and higher photostability to avoid photobleaching. All
10
these types of plasmonic NPs can be prepared with their SPR peaks tuned to match
the center wavelength of laser irradiation for maximal absorbance, which should be
located in the NIR window (650–900nm)130 where light penetration is optimal due to
minimal absorption by water and hemoglobin in the tissue. Compared to other types
of nanoscale photothermal absorbers (e.g., carbon nanotubes131 and TiO2
nanotubes132), the plasmonic NPs have advantages on dual imaging/therapy functions
(see Section 1.4.2).
1.4.4 Stimuli-sensitive drug delivery agents
Nanomaterials that are sensitive to external stimuli are of emerging interest due to
their great potential in many biomedical and technical applications. For instance,
significant efforts have been devoted to the development of “smart” delivery systems
in which uptake and delivery of molecules can be controlled by a variety of external
stimuli, e.g. temperature,133, 134 pH value,43, 135 magnetic field,11 electric field,136
photoirradiation,18 ultrasound,19 biomolecules,20 and mechanical forces.137 The
previously discussed PDT is actually a stimuli-responsive system where the light of
specific wavelengths is used to trigger the therapeutic activity (see Section 1.4.3).
Among the environmentally sensitive nanomaterials, temperature-responsive
materials are probably one of the most commonly studied classes,138 and the idea
naturally came from the fact that many pathological areas demonstrate distinct
hyperthermia. Temperature-sensitive polymers of PNIPAAm and derivatives are the
most extensively used, which undergoes temperature-responsive conformational
change from the swelled, hydrophilic state to the shrunken, hydrophobic state when
above its LCST of 32-33 ˚C which is close to physiological temperature.87, 139, 140
Various kinds of PEGylated liposomes have been prepared for pH-sensitive drug
delivery,141, 142 which undergo degradation to release the contents upon exposure to
lowered pH in pathological sites such as tumors, infarcts, or inflammation zones.143-145
Functional Materials Division, KTH, 2011 11
1.5 Biomedical applications of nanomaterials
1.5.1 Magnetic resonance imaging
1.5.1.1 Physical background
The basic principle of MRI is based on NMR which is the interaction between
magnetic field and protons. Under a given external magnetic field (B0, T), protons
experience a torque, since they cannot align exactly with the external field, which
make them precess around the direction of the field. The precessional frequency (ω0,
MHz) of the protons is found to be proportional to the external magnetic field, given
by the Larmor equation:
00 Bγω =
where γ is the gyromagnetic ratio (γ = 42.57 MHz T-1 for 1H), i.e. the ratio of the
magnetic dipole moment of a particle to its angular momentum. All the protons in a
magnetic field precess at the same Larmor frequency, which is a resonance condition.
Dependent on its internal energy, proton will precess in one of only two orientations,
either spin-up or spin-down.
When a resonant RF transverse pulse is perpendicularly applied to B0, it causes
resonant excitation of the magnetic moment precession into the perpendicular plane.
Upon removal of the RF, the magnetic moment gradually relaxes to equilibrium by
realigning to B0. Such relaxation processes involve two pathways:
a) longitudinal or T1 relaxation accompanying loss of energy from the excited
state to its surroundings (lattice) and recovery of decreased net magnetization (Mz) to
the initial state, and
b) transverse or T2 relaxation from the disappearance of induced magnetization on
the perpendicular plane (Mxy) by the dephasing of the spins.
These processes can be expressed as follows.
)1( 1Tt
z eMM−
−= (longitudinal)
12
2)sin( 0T
t
xy etMM−
Φ+= ω (transverse)
where T1 and T2 are the longitudinal and transverse relaxation time, respectively. Such
relaxation processes are recorded and then reconstructed by MRI to obtain gray scale
images.
1.5.1.2 Contrast agents
Biological tissues and organs have aqueous environments (70%-90% water in
most tissues) that vary in density and homogeneity, which are imaged as having
different contrast (signal) and provide anatomical information. Contrast agents help to
improve the specificity of MR imaging by producing an extra set of images with
different contrast, as a result of the interaction between the induced local magnetic
field and the neighboring water protons. The signal intensity of MRI primarily
depends on the local values of longitudinal (1/T1) or transverse (1/T2) relaxation rate
of water protons. The relaxivities of r1 and r2, which are commonly expressed in
mM-1 s-1, indicate respectively the increase in 1/T1 and 1/T2 per concentration [M] of
contrast agent:
][11
0,
MrTT i
ii
+= (i = 1, 2)
where Ti,0 is the relaxation times of native tissues (i.e., tissue devoid of exogenous
contrast agent). The efficiency of an MRI contrast agent is assessed in terms of the
ratio between the transverse and longitudinal relaxivities (expressed as r2/r1), which is
a defining parameter indicating whether the contrast agent can be employed as a
positive (T1) or negative (T2) agent. Commercially available T1 contrast agents are
usually paramagnetic complexes, e.g. Gd-DOTA (DotaremTM), Gd-DTPA-BMA
(OmniscanTM), and Mn-DPDP (TeslascanTM), while T2 contrast agents are based on
SPIONs, e.g. FeridexTM, ResovistTM, and CombidexTM. Due to some limitations of
these commercial contrast agents, such as polydispersity, poor crystallinity and
magnetic properties, new generation of contrast agents have being developed to
improve MR contrast effects with a less amount of dosage.
Functional Materials Division, KTH, 2011 13
1.5.1.3 Superparamagnetism
An important requirement for useful T2 contrast agents is a large r2/r1 ratio, in
combination with high absolute value of r2. This condition can be achieved for agents
with predominantly outer sphere relaxation mechanism.146 Contrast agents that satisfy
these conditions are SPIONs. Another important parameter used to characterize ferri-
or ferromagnetic materials is anisotropy energy (Ea):
VKE aa =
where Ka is the anisotropy constant and V the volume of the crystal. Anisotropy
energy is the variation amplitude of magnetic energy of a nanomagnet dependent on
the direction of its magnetization vector.147 For the colloid of ferromagnetic crystals,
the return of the magnetization to equilibrium is determined by two different
processes, Néel relaxation and Brownian relaxation. When the magnetization curve is
perfectly reversible, because of thermodynamic equilibrium of system by fast
magnetic relaxation, the behavior has been named “superparamagnetism” by Bean
and Livingston.148 When the anisotropy energy of magnetic particles is larger than the
thermal energy kBT, where kB is the Boltzmann constant and T the absolute
temperature, i.e. TkEa B>> , the direction of magnetic moment maintains very close
to that of the anisotropy axes. In small crystals, the anisotropy energy is comparable
to the thermal energy, i.e. TkEa B≤ , so that the magnetic moment is no longer fixed
along the easy directions, which allows superparamagnetic relaxation. SPIONs can
cause noticeable shortening of T2 relaxation times with signal loss in the targeted
tissue and generate contrast to other tissues.
1.5.1.4 Preparation methods for magnetic NPs
Numerous chemical methods have been developed to synthesize magnetic NPs
for biomedical applications. There are some concerns for the synthetic methods
including development of a reproducible process without any complicated purification
14
procedure and experimental conditions leading to monodisperse magnetic grains with
suitable size.
Coprecipitation, the most widely used method, is probably the simplest and most
efficient chemical pathway to obtain magnetic NPs, where the soluble precursors
precipitate upon the addition of anionic counter ions. Iron oxides, either Fe3O4 or
γ-Fe2O3, can be prepared from aqueous Fe2+/Fe3+ salt solutions by the addition of a
base under inert atmosphere at room temperature or at elevated temperature.149 The
chemical reaction of Fe3O4 formation can be written as:
OHOFeOHFeFe 24332 482 +→++ −++
This method is also applied for synthesis of spinel-type ferromagnets, such as
MnFe2O4,150 ZnFe2O4,151 and CoFe2O4.152 The size, shape, and composition of the
magnetic NPs are dependent on the type of salts used (e.g. chlorides, sulfates, nitrates),
the Fe2+/Fe3+ ratio, the reaction temperature, the pH value, and ionic strength of the
media. The advantages of coprecipitation method are high reproducibility of the NPs
and scalable yield, while the shortcoming is weak shape control and broad size
distribution. Efforts have been made in preparing monodisperse magnetite NPs.153
Thermo-decomposition method has been developed as an effect way to synthesize
high-quality semiconductor and oxide NPs with controlled size and shape,154, 155 in
which organometallic compounds as precursors are decomposed in high-boiling
non-aqueous media containing stabilizing surfactants.156, 157 The organometallic
precursors include metal acetylacetonates,154 metal-oleate complex,155 metal
cupferronates,158 or carbonyls.159 Fatty acids,160 oleic acid,161 and hexadecylamine162
are often used as surfactants. The separation of nucleation and aging period during the
particle formation allow the production of highly crystalline and monodisperse
magnetic NPs with selected particles sizes.
Other methods, including microemulsion,163 hydrothermal,164 and sol-gel,165 are
also investigated for synthesis of magnetic NPs. Several phase transfer strategies29, 166,
167 have been applied to hydrophobic magnetic NPs for obtaining their aqueous
dispersion.
Functional Materials Division, KTH, 2011 15
1.5.2 Plasmonic photothermal therapy
In photothermal ablation, optical irradiation is absorbed and transformed into heat,
inducing thermal denaturing of proteins and DNA in the cells, consequently causing
irreversible damage to the targeted tissue.168 It is desirable to use agents that are active
in the NIR region of the radiation spectrum to minimize the light extinction by
intrinsic chromophores in native tissue.130 Noble metal NPs have been used for the
application of photothermal cancer therapy,111 due to their strong electric fields at the
surface to enhance the absorption and scattering of electromagnetic radiation.
1.5.2.1 Surface plasmon resonance
Surface plasmons are surface electromagnetic waves that propagate in a direction
parallel to the metal/dielectric (or metal/vacuum) interface. The critical conditions for
existence of surface plasma are that the real part of the dielectric constant of the metal
must be negative and its magnitude must be greater than that of the dielectric, which
can be met in the IR-visible wavelength region for air/metal and water/metal
interfaces. The maximum and bandwidth of surface plasmon band are also influenced
by the particle shape, medium dielectric constant, and temperature. When plasmonic
NPs (gold or silver) are exposed to laser light resonant with their surface plasmon
oscillation, they can strongly absorb the light and rapidly convert it into heat via a
series of photophysical processes.169, 170
1.5.2.2 Preparation methods for gold NPs and NRs
In 1857, Faraday reported the formation of deep-red solutions of colloidal gold by
reduction of an aqueous solution of chloroaurate (AuCl4-) using phosphorus in CS2 (a
two-phase system) in a well-known work.171 Nowadays, various methods for the
preparation of gold colloids were reported and reviewed, after the breakthrough of
two classical works as citrate reduction method by Turkevitch172 and two-phase
method by Brust.173 Later, microemulsion,174 seeding growth,175 thermolysis176
16
methods were developed for synthesis of Au NPs. A popular method for growth of Au
nanorods is seed-mediated protocol originated by the work of Murphy et al.,177 in
which TSC-capped small Au NPs were added as seeds into growth solution containing
gold salt and surfactant for further growth of Au nanorods. Other methods include
earlier photochemical reduction,178 electrochemical,179 template,180 and top-down
lithographic methods.181
1.5.3 Temperature-triggered drug release
The driving force for the extensive studies on temperature-sensitive materials in
biomedicine is their potential for precise, on-demand content delivery in vitro or in
vivo. The controlled release of payload from a variety of carriers can be triggered by
illuminating with light at resonant wavelengths to enable photothermal conversion 182
or receiving thermal energy from the environment.37 Thermo-sensitive polymers are
one of the intensively studied materials for controlled drug release in combination
with proper drug carriers to form copolymers133 or core-shell structures.183
1.5.3.1 Thermo-responsive phase transition
PNIPAAm is a temperature-responsive polymer that was first synthesized in the
1950s.184, 185 In dilute aqueous solution, single chains of PNIPAAm undergoe a
coil-to-globule transition when temperature is raised from below LCST to above
LCST, where isolated, flexible but extended coil of polymer chain becomes collapse
and aggregation after the transition. When cross-linked, the hydrogel of PNIPAAm
shows a reversible LCST phase transition from a swollen hydrated state to a shrunken
dehydrated state, losing about 90% of its mass. Studies on gel transition theory show
that both hydrogen bonding and hydrophobic effect contribute to the driving force for
the transition, reflecting the changes in free energy.186, 187
Functional Materials Division, KTH, 2011 17
1.5.3.2 Preparation methods for thermo-sensitive polymers
PNIPAAM has been synthesized mainly by the radical polymerization of
NIPAAm via a variety of techniques including the typical free radical initiation of
organic solutions188 and redox initiation in aqueous media.189 Other synthetic schemes
such as using ionic initiators190, 191 and radiation polymerization192, 193 have also been
investigated. Later, living polymerization methods using free radical chemistry, e.g.
RAFT133 and ATRP,194 have been developed to obtain controlled molecular weight
polymers with narrow polydispersity indexes and various complex architectures.133, 195,
196
18
2 Experimental
2.1 Magnetic mesoporous NPs
2.1.1 Synthesis of SPION and phase transfer
Monodisperse iron oxide NPs were synthesized using previously reported
method155 via thermal-decomposition of Fe-oleic complex in dioctyl ether at ca. 297
°C in the presence of oleic acid. Oleic acid capped Fe3O4 NPs were transferred to
water by mixing with aqueous CTAB solution at 65 ˚C and the emulsion was
vigorously stirred until all the chloroform was evaporated.
2.1.2 Fabrication of Fe3O4@mSiO2
The procedure of growing mSiO2 on CTAB capped Fe3O4 NPs is schematically
depicted in Figure 2.1. The aqueous Fe3O4 suspension was diluted with certain
amount of water and the pH was tuned to 12 by the addition of NaOH. The solution
was heated and, when the temperature of the solution reached 70˚C, a specific amount
of TEOS and corresponding four times of EtOAc were slowly added to the reaction
solution in sequence. After the reaction was conducted for 2 h, the core-shell particles
were collected by centrifugation and washed by EtOH two times. To extract CTAB
from the channels of mSiO2, the particles were dispersed in ethanolic NH4NO3
solution (10 mg/mL) at 60˚C and mixed for 30 min.197 After washing twice with
EtOH, the Fe3O4@mSiO2 NPs were re-dispersed in water for further use.
Figure 2.1 Synthetic diagrams of preparation of Fe3O4@mSiO2 core-shell NPs.
Functional Materials Division, KTH, 2011 19
2.2 Preparation of thermo-sensitive composite NPs
The coating procedure of copolymer is schematically illustrated in Figure 2.2.
Prior to grafting of copolymer, Fe3O4@mSiO2 NPs without extraction of CTAB were
functionalized with carbon–carbon double bonds by reacting with TMSMA under
refluxing and N2 purging. The TMSMA-modified Fe3O4@mSiO2 NPs were used as
the seeds to conduct free radical copolymerization of NIPAAm and AAm on the NPs.
Typically, monomers of NIPAAm and AAm and cross-linker MBA were mixed with
aqueous suspension of TMSMA-modified Fe3O4@mSiO2 NPs at 70 ˚C under a N2
flow before the initiator potassium persulfate (KPS) was introduced. The composite
NPs were then collected and washed by water to remove the unreacted monomers.
Figure 2.2 Synthesis procedure for preparation of core-shell composite Fe3O4@mSiO2@P(NIPAAm-co-AAm) NPs.
2.3 Synthesis of gold nanorods
In the present work, high AR gold nanorods were prepared by reducing the
growth solution of HAuCl4 and CTAB with the addition of HNO3 under ambient
temperature. A weak reducing agent, ascorbic acid, was used to reduce Au3+ to Au+
and a strong reducing agent of NaBH4 induced the formation of gold nanorods. Before
in vitro experiments, high AR gold nanorods were treated by a series of purification
steps to ensure the removal of CTAB from their surfaces but without causing
agglomeration. The procedure for synthesis of low AR gold nanorods is similar with
that for high AR ones with the only exception of using AgNO3 instead of HNO3 to
restrict the anisotropic growth of gold nanorods.198
20
2.4 Characterizations
The morphology of NPs was characterized by JEM-2100F field emission TEM
operating at an accelerating voltage of 200 kV. Powder XRD patterns were recorded
on a PANalytical XPert Pro powder diffractometer with Cu-Kα radiation (45 kV, 35
mA). Nitrogen sorption isotherms were obtained on a Micromeritics ASAP 2020 pore
analyzer at 77 K under continuous adsorption conditions. The BET equation was used
to calculate the surface area from adsorption data obtained in the range of relative
pressure p/p0 = 0.05 and 0.3. The total pore volume was calculated from the amount
of N2 adsorbed at p/p0 = 0.98. NLDFT model was applied to determine the PSD. The
hydrodynamic diameter of the particles was measured by PCS (DelsaTMNano particle
size analyzer, Beckman Coulter). The surface charge of NPs in suspension was
evaluated by Malvern Zetasizer. Concentrations of Fe or Au were measured by
Thermo Scientific iCAP 6500 series ICP-AES and Varian AAS, respectively. The
thermal behavior of composite NPs was measured DSC (Q2000, TA instruments). The
magnetization measurements were performed using a VSM (NUOVO MOLSPIN,
Newcastle Upon Tyne, Great Britain). The longitudinal NMRD profiles were recorded
at 37 °C on a Fast Field Cycling Relaxometer (Stelar, Italy) and the magnetic field
ranges from 0.013 MHz to 60 MHz. The radius of the magnetic particle r, which
determines the distance of closest approach between the paramagnetic center and the
water molecule, is determined by τD=r2/D from relaxometry data, where ωI is the
angular frequency of the proton precession, D is the relative diffusion coefficient
between the paramagnetic center and the water molecule. The specific magnetization
Ms relaxo can be obtained from Ms relaxo~(Rmax/CτD)1/2, where C is a constant and Rmax
the maximal relaxation rate in NMRD. Additional relaxivity r1 (20 and 60 MHz) was
measured at 0.47 T with a Minispec PC-20 Bruker spectrometer and 1.41 T with Mq
Series systems; r2 was measured with Minispec (20 and 60 MHz) on spectrometer
AMX-300.
Functional Materials Division, KTH, 2011 21
2.5 Biocompatibility evaluation
2.5.1 Co-culture immature MDDCs with gold nanorods
Human peripheral blood mononuclear cells (PBMC) from healthy blood donors
were separated from buffy coats by standard Ficoll gradient centrifugation. MDDCs
were generated in the presence of IL-4 and GM-CSF as previously described.199 At
day 6 of culture, the immature MDDCs (4×105 cells/mL) were co-cultured with gold
nanorods at a concentration of 0.5, 5 or 50 µg/mL for 24 h. As controls, MDDCs
cultured in medium alone or with LPS (0.1 µg/mL; L8274, Escherichia coli, serotype
026-B6) were used. The morphology of MDDCs incubated with gold nanorods were
examined by Tecnai 10 TEM at 80 kV.
2.5.2 Flow cytometry analysis
The phenotype of MDDCs was evaluated with flow cytometry analysis. MDDCs
were labeled with fluorescent phycoerythrin (PE) conjugated mouse monoclonal
antibodies (mAbs) specific for: CD1a and CD11c; and with fluorescein isothiocyanate
(FITC) conjugated mAbs specific for: CD40, CD80, CD83, CD86 and HLA-ABC,
HLA-DR and CD14 according to the manufacturer’s instructions. Control samples
were labeled with isotype-matched antibodies conjugated with the same fluorochrome.
Fluorescence was measured with a FACSCalibur flow cytometer. Ten thousand cells
were counted and analyzed by the program CellQuestPro.
2.5.3 Cell viability assay
To investigate the effect of gold nanorods on MDDCs’ viability, cells were
examined for the degree of apoptosis and necrosis by measuring the binding of
Annexin V-fluorescein and inclusion/exclusion of propidium iodide (PI) using a
FACSCalibur flow cytometer. PI-/Annexin V+ events defined early apoptosis whereas
PI+/Annexin V+ signals were counted as late apoptosis or secondary necrosis.
22
3 Results and discussions
3.1 Superparamagnetic mesoporous core-shell NPs
3.1.1 Morphology study
Figure 3.1a shows the TEM micrograph of oleic acid capped SPIONs dispersed in
chloroform with narrow size distribution and an average diameter of 10.9 nm (σ ≈ 5.0
%). High resolution TEM image shows the single crystalline nature of the particles
(inset of Figure 3.1a). After phase transfer, aqueous SPIONs capped by CTAB
retained its morphology. Fe3O4@mSiO2 NPs with diameters varying from ca. 25 nm
to ca. 95 nm were synthesized. The low magnification images display that all the
particles are uniform and separated from each other (Figure 3.1). The insets of high
magnification images for all the core-shell NPs show a mSiO2 layer with about 2 nm
wormhole-like mesopores. We investigated the influence of reagents used for
Figure 3.1 TEM micrographs of (a) hydrophobic Fe3O4 core NPs, and core–shell NPs of (b) 50 nm, (c) 75 nm, and (d) 95 nm Fe3O4@mSiO2 with the same core. The insets show the magnified images for each sample.
Functional Materials Division, KTH, 2011 23
synthesis of mSiO2 coatings and the optimized parameters for producing well-defined
core-shell structures are summarized in Table 3.1. Desired concentrations of CTAB
were chosen to assist phase transfer of hydrophobic magnetite NPs to aqueous phase,
and are responsible for the thickness of subsequent mSiO2 coating layer. For certain
amount of core materials, an excess amount of CTAB induces extra mSiO2 spheres
without SPION in the cores. On the other hand, deficiency of CTAB limits the shell
thickness despite the excessive amount of TEOS available. In the optimized system
with a specific ratio between Fe3O4, CTAB and water, where mSiO2 forms the coating
layer a single magnetite core, an excess amount of TEOS will not induce extra
core-free mSiO2 spheres, instead that TEOS condenses in the pores leading to pore
blockage. It is shown in Table 3.1 that, to grow thicker mSiO2 shell, the
[CTAB]/[Fe3O4] and [H2O]/[CTAB] have to increase to allow more TEOS to be
hydrolyzed and condenses around the magnetic core. The addition of EtOAc was
found to be crucial to produce well separated single-cored core-shell NPs.
Table 3.1 Summary of reactants composition for synthesis of Fe3O4@mSiO2 NPs with different particle sizes.
[CTAB]/[Fe3O4]a
Molar compositionb
Average particle
diameter, nmc 11.7 1 CTAB: 4.87 TEOS: 1.08 NaOH: 5983 H2O 25 11.7 1 CTAB: 9.74 TEOS: 2.15 NaOH: 11966 H2O 35 35.1 1 CTAB: 6.50 TEOS: 0.72 NaOH: 3989 H2O 45 35.1 1 CTAB: 9.74 TEOS: 1.44 NaOH: 7977 H2O 55 46.8 1 CTAB: 5.41 TEOS: 0.72 NaOH: 3989 H2O 65 70.2 1 CTAB: 8.12 TEOS: 1.08 NaOH: 5983 H2O 75 70.2 1 CTAB: 12.2 TEOS: 1.80 NaOH: 9972 H2O 85 93.6 1 CTAB: 12.2 TEOS: 2.02 NaOH: 11218 H2O 95
a 0.5 mL of Fe3O4 NPs suspension in CHCl3 (7.9 mg Fe/mL) was conducted for phase transfer; b CTAB refers to as the [CTAB] used previously for phase transfer; c particle diameters of Fe3O4@mSiO2 were determined by TEM (n=200).
24
3.1.2 Characterization of mesoporous silica
Small angle XRD pattern of Fe3O4@mSiO2 (Figure 3.2a) exhibits a characteristic
mesoporous structure with a reduction in the long-range mesoscale order indicated by
the absence of high order reflections.200 In Figure 3.2b, the N2 adsorption/desorption
isotherms exhibit a characteristic type IV isotherm as expected for mSiO2. The
corresponding pore size distribution (PSD) calculated by NLDFT model (inset of
Figure 3.2b) demonstrates a bimodal porosity (pore size 14 Å and 27 Å), which might
be induced by the heterogeneous distribution of magnetite NPs in the porous network
and hence a broad distribution of PSD. The BET surface area of 75 nm Fe3O4@mSiO2
NPs is 202 m2 g-1 and the total pore volume is 0.29 cm3 g-1.
Figure 3.2 Characterization of extracted Fe3O4@mSiO2 NPs dried at 80 ˚C, (a) small angle XRD pattern, (b) N2 adsorption (solid)-desorption (hollow) isotherms (inset: NLDFT slit type model for PSD from adsorption branch).
In wide angle XRD pattern, the broadest peak centered at 22˚ (2θ) (Figure 3.3b) is
consistent with the amorphous nature of the silica wall and the characteristic peaks of
a magnetite structure indicates that the magnetic core embedded in silica retains its
original crystalline nature after mSiO2 coating and surfactant extraction. The average
size of the Fe3O4 cores was calculated by using the Debye–Scherrer formula and is
determined as 10.5 nm, which is in good agreement with the average diameter
measured from TEM images, indicating they are dominantly single crystalline.
Functional Materials Division, KTH, 2011 25
Figure 3.3 The powder X-ray diffraction (XRD) of (a) Fe3O4 and (b) Fe3O4@mSiO2.
3.1.3 Magnetometric and relaxometric properties
Field dependent magnetism of CATB-capped Fe3O4 and Fe3O4@mSiO2 NPs with
different shell thicknesses was examined at room temperature by using VSM. None of
the samples showed a hysteresis (Figure 3.4a), demonstrating that they are
superparamagnetic, which is a desirable characteristic for T2 MR contrast agents. The
saturated magnetization (Ms) of Fe3O4-CTAB, 50 nm and 75 nm Fe3O4@mSiO2 NPs
are 48.7, 48.3, and 47.1 emu/g Fe, respectively. Since Ms determines the magnetic
moment and a higher value indicates a higher magnetic susceptibility, i.e. stronger
MRI signals,201 Fe3O4@mSiO2 NPs show implications of high MRI sensitivity. Fitting
the magnetization data to Langevin equation,148 we obtained the magnetic domain size
of the magnetic cores of these samples as 11.7, 11.8, and 11.8 nm respectively, which
Figure 3.4 (a) Field-dependent magnetization measurement of aqueous suspensions of CTAB-capped Fe3O4 and Fe3O4@mSiO2 (50 nm and 75 nm, repectively) NPs at room temperature, (b) NMRD profiles of CTAB-capped Fe3O4 and 50 nm Fe3O4@mSiO2 NPs recorded at 37 ˚C.
26
are close to the values obtained from TEM and XRD. On the other hand, the NMR
relaxometry plays an important role in evaluating the properties of the
superparamagnetic colloids as potential contrast agents.202 The effects of surface
coatings on the relaxometric properties of SPIONs are examined and CTAB-capped
and mSiO2-capped Fe3O4 NPs are compared through the characterization by NMRD
profiles (Figure 3.4b). By fitting the NMRD profiles with adequate theories,203, 204 the
average diameter of Fe3O4-CTAB is 12.5 nm and specific magnetization 48.2 emu g-1,
and τN 1.25×10-9 s, which are highly coherent with the results of magnetic domain
size and Ms by magnetometry. The NMRD of 50 nm-sized Fe3O4@mSiO2 provide the
diameter of the magnetite core embedded in mSiO2 as 28 nm, specific magnetization
as 14.3 emu g-1, and τN 5.85×10-9 s, which are much different from the results
obtained by magnetometry. This may be due to the effect of diffusive permeability of
Fe3O4@mSiO2 to water molecules, since the determination of magnetic crystal radius
r and specific magnetization Ms relaxo by NMRD are closely related with diffusion
parameters (e.g., τD and D).204, 205
3.1.4 MR studies of Fe3O4@mSiO2 NPs
The r1 and r2 relaxivity values and hydrodynamic sizes of Fe3O4-CTAB and
Fe3O4@mSiO2 with various coating thicknesses are summarized in Table 3.2.
Fe3O4-CTAB shows similar value of r2 with those of Fe3O4@mSiO2 NPs, however the
value of r1 is one magnitude higher than those of Fe3O4@mSiO2. Since the effects of
surface chemistry on relaxivity most likely arises from the hydrophilicity of the
coating layers and the coordination between the inner capping ligands, both
influencing spin orders,206 the variation of r1 is proposed to be due to the different
hierarchical structures between CTAB and mSiO2 layer around the magnetite core.
For Fe3O4@mSiO2 with varied coating thickness, it is found that the values of r1 are
effectively decreased by ca. 6 times (at 20 MHz) or ca. 4.5 times (at 60 MHz) when
particle size increases from 50 nm to 95 nm, whilst r2 is decreased by only ca. 40 % at
Functional Materials Division, KTH, 2011 27
20 and 60 MHz. The magnitude of r1 is reported to be dependent on the magnetization
of the material, the electron spin relaxation time, and the accessibility of protons
bearing nuclear spins to the surface of iron oxide.207 The decreased value of r1 for
Fe3O4@mSiO2 with increased silica shell thickness may reflect the ability of mSiO2
coatings on separating water from the surface of the magnetite NPs, which is critical
for r1 values.208 Similar surface chemistry studies on relaxivity of polymer coated
Fe3O4 also showed that the hydrophilicity of the coating layer has a positive effect on
enhancing the proton relaxivities (both r1 and r2).167, 206 Superparamagnetic T2 contrast
agents were known to be able to produce a long-range magnetic field to promote the
spin-spin relaxation process of surrounding water molecules,23 and the magnitude of
r2 is believed to reflect the ability of the magnetic material to produce local
inhomogeneity in the magnetic field.209 Our results suggest that the locally generated
magnetic field by Fe3O4 cores has been weakened by the increased thickness of
mSiO2 coating.
Considering the NMRD data and hydrodynamic size measurement,
Fe3O4@mSiO2 NPs are assumed consisting of three regions: iron oxide core
generating a magnetic field, water impermeable layer with radius of rim, and slow
diffusion layer of protons with radius of rdiff (Scheme 3.1a). In Scheme 3.1b, thicker
Table 3.2 Mean hydrodynamic diameters and relaxivities (r1 and r2 are in unit of [s-1 mM-1]) of CTAB-capped Fe3O4 and different sized Fe3O4@mSiO2 NPs measured at 20 MHz (0.47 T) and 60 MHz (1.41 T) in water (37 °C), and the reported relaxivity values for commercial Feridex® and Resovist® contrast agents.
sample name mean 20 MHz 60 MHz
(particle diameter hydrodynamic
by TEM) diameter [nm]a
r1
r2
r2/r1
r1
r2
r2/r1
Fe3O4–CTAB (11 nm) 67 31.25 81.37 2.61 13.69 82.18 6.01Fe3O4@mSiO2 (50 nm) 60 3.65 84.26 23.1 1.31 92.13 70.3Fe3O4@mSiO2 (75 nm) 77 2.13 79.93 37.5 0.97 87.54 90.3Fe3O4@mSiO2 (95 nm) 96 0.61 50.13 82.2 0.31 55.44 179 Feridex208 72 40 160 4 − − − Resovist210 65 25 164 6.2 − − − a mean value of volume distribution
28
Scheme 3.1 Schematic representations of (a) a Fe3O4@mSiO2 NP labeled with rim (radius of water impermeable region indicated by dashed circle) and rdiff (radius of slow proton diffusion region indicated by dotted circle), (b) water diffusions in the coating zones with different thickness under magnetic field.
mSiO2 coated Fe3O4@mSiO2 has more water impermeable part than the thinner
coated one, therefore excludes more water and the relaxation rate of protons in
diffusion layer is slower, together resulting in the decrease of r2.
Owing to a higher impact of mSiO2 coatings on r1 than on r2, the r2/r1 ratio
increases as a function of the coating thickness, which are 82.2 and 179 at 20 MHz
and 60 MHz for 95 nm Fe3O4@mSiO2, respectively. These great enhancement of r2/r1
ratios, ca. 21 and ca. 14 times higher than the two commercial iron-oxide-based
contrast agents (see Table 3.2), indicates Fe3O4@mSiO2 a high efficiency on T2 MR
imaging. By comparison, in previous reports on relaxivity variation of conventional
amorphous SiO2 coated Fe3O4, there is no enhancement for r2/r1 ratios211, 212 when the
thickness of SiO2 coatings increases. The differences on the enhancement of r2/r1
ratios between conventional amorphous SiO2 and mSiO2 are most probably due to
their different properties on water permeability.
3.2 Multifunctional core-shell composite NPs
3.2.1 Morphological and structural studies
Figure 3.5 shows the TEM images of poly(NIPAAm-co-AAm) coated
Fe3O4@mSiO2 NPs with different thicknesses of polymer layers. The thickness of
polymer coating was ca. 10 nm (Figure 3.5b) when 3 mmole of monomers were used,
Functional Materials Division, KTH, 2011 29
Figure 3.5 TEM micrographs of (a) 85 nm Fe3O4@mSiO2 core–shell NPs, P(NIPAAm-co-AAm) coated 85 nm Fe3O4@mSiO2 composite NPs with different thicknesses of polymer layers using (b) 3 mmole, and (c) 12 mmole of NIPAAm and AAm monomers. The insets show the magnified images for each sample, and the polymer layer was visualized after positive stain with phosphotungstic acid
and it increased to ca. 30 nm (Figure 3.5c) when the amount of monomers increased
to 12 mmole. The observed necking of thick polymer coatings in Figure 3.5c is due to
a relatively high concentration of monomers and cross-linker. The thick polymer
coatings are reported to have less obvious hydrophilic–hydrophobic transition,
probably due to the existence of less flexible polymer chains.213 Hence, a thin layer of
polymer coatings is desired for thermo-responsive applications and to avoid blocking
the mesopores.
N2 adsorption/desorption isotherm (Figure 3.6a) of Fe3O4@mSiO2@P(NIPAAm-
co-AAm) NPs exhibit a characteristic type IV isotherm for mSiO2 structures. The pore
Figure 3.6 (a) N2 adsorption (solid)-desorption (hollow) isotherms, (b) PSD of extracted Fe3O4@mSiO2 (square) dried at 80 ˚C and Fe3O4@mSiO2
@P(NIPAAm-co-AAm) freeze dried at -78 ˚C (triangle).
30
size is calculated as 14 Å and 17 Å (a bimodal porosity). The BET surface area is 30.4
m2 g-1 and total pore volume is 0.15 cm3 g-1. A similar decrease of surface area and
pore volume after polymer grafting on mSiO2 was previously reported,134 where 19 %
decrease for heating-dried and 72 % decrease for freeze-dried thermo-sensitive
polymer coated NPs were observed.
3.2.2 Magnetic properties
Magnetic measurement of Fe3O4-CTAB, 85 nm Fe3O4@mSiO2, and
Fe3O4@mSiO2@P(NIPAAm-co-AAm) NPs was perform using VSM. None of the
samples showed a hysteresis (Figure 3.7), demonstrating that all of them are
superparamagnetic. The magnetic domain size of the magnetic cores of these samples
are obtained as 10.6, 10.4, 9.9 nm respectively according to Langevin equation.148
These values are comparable with the size estimated from TEM micrographs. The Ms
of CTAB-capped Fe3O4 is 57.2 emu g-1 Fe. For 85 nm Fe3O4@mSiO2 NPs, Ms is 52.7
emu g-1 Fe or 1.59 emu g-1 for Fe3O4@mSiO2 (ca. 3.0 wt % of Fe in Fe3O4@mSiO2).
The polymer-coated composite, prepared from a total of 3 mmol of NIPAAm and
AAm monomers (90%:10% in molar ratio), shows Ms of 49.1 emu g-1 Fe or 1.13 emu
g-1 for composite (ca. 2.3 wt % of Fe in composite). The T2-enhancing capability of
mSiO2 and polymer coated magnetite NPs is measured at 0.47 T, and r2 is obtained as
164 s-1 mM-1 and 113 s-1 mM-1, respectively.
Figure 3.7 Field-dependent magnetization measurement of Fe3O4 (■), Fe3O4@mSiO2
(○), and Fe3O4@mSiO2@P(NIPAAm-co-AAm) (△) at room temperature.
Functional Materials Division, KTH, 2011 31
3.2.3 Thermo-responsive properties and manipulation of LCST
The colloidal properties of the Fe3O4@mSiO2@PNIPAAm NPs, prepared from 3
mmol of NIPAAm monomers, were studied by measuring the hydrodynamic particle
sizes. As shown in Figure 3.8a, the composite NPs maintain their initial size (ca. 200
nm, mean value of volume distribution) over a week, which indicates that they are
stable in phosphate buffered saline (PBS). The temperature-dependent hydrodynamic
size (Figure 3.8b) of the composite NPs were measured to show their
thermo-sensitivity. In Figure 3.8b, at 20 °C the particle size is 211 nm, and a sharp
decrease of the particle size is observed from 29 °C to 37 °C due to the collapse of the
PNIPAAm chain. The particle size is 144 nm at 37 °C and almost no change is
observed till 45 °C. By fitting the variation of hydrodynamic size on temperature
change, phase transition temperature, i.e. LCST, of the polymer coated composite NPs
is found at ca. 33 °C.
The thermo-responsive properties of the Fe3O4@mSiO2@P(NIPAAm-co-AAm)
NPs and the LCST of the polymer shell were further studied utilizing DSC. An
aqueous suspension of PNIPAAm coated Fe3O4@mSiO2 NPs using 1.5 mmol
NIPAAm was observed to exhibit an endothermic peak at 36.9 ˚C in heating process
and an exothermic peak at 34.7 ˚C in cooling process (Figure 3.9a). Such hysteresis of
phase transition may be due to kinetic effects. When the amount of monomer of
NIPAAm increases to 3 mmol, a similar endothermic peak of polymer coated NPs was
Figure 3.8 Hydrodynamic diameters (average mean values of volume distribution ± standard deviation) of Fe3O4@mSiO2@PNIPAAm NPs in PBS (a) at 25 °C for different temporal points, (b) versus temperature
32
Figure 3.9 DSC curves of Fe3O4@mSiO2@PNIPAAm samples show phase transitions and the variation of transition temperatures with the increase of monomer amount from (a) 1.5 mmol to (b) 3 mmol of NIPAAm in three or five continuous heating–cooling cycles, respectively. found at 34.8 ˚C and an exothermic peak at 32.8 ˚C (Figure 3.9b). Both of the
polymer coatings show a good reversibility of the phase transition demonstrated by
three or five continuous heating–cooling cycles, respectively. When the cross-linking
density is fixed, i.e. 10 % weight percent of MBA in monomers, LCST is decreased
for higher NIPAAm content polymer shell than the lower one. This is consistent with
the previous reports that as the polymer chain contains more hydrophobic constituent,
LCST becomes lower.214 Compared with the LCST determined by DLS as 33 °C, the
lag of response in DSC measurement showing endothermic peak at 34.8 ˚C might be
due to the slow equilibration of the temperature in the measuring cell.
On the other hand, LCST can also be manipulated by adjusting the ratio of
hydrophobic and hydrophilic segment of the polymer. In the present work, we
introduce a hydrophilic co-monomer of AAm to produce copolymer with NIPAAm. A
total of 3 mmole of NIPAAm and AAm with varied molar ratios were applied to coat
the same amount of Fe3O4@mSiO2 NPs as PNIPAAm coated ones with the
cross-linking density unchanged. Figure 3.10 shows that the endothermic peaks of 5
%, 10 %, and 15 % AAm content of the co-polymer coatings are located at 37.6 ˚C,
40.1 ˚C, and 42.4 ˚C, respectively, while the exothermic peaks are at 35.3 ˚C, 37.6 ˚C,
and 39.6 ˚C, respectively. All samples with different compositions showed reversible
phase transition behavior in five continuous heating-cooling cycles. We found that
Functional Materials Division, KTH, 2011 33
Figure 3.10 DSC curves of Fe3O4@mSiO2@P(NIPAAm-co-AAm) samples show phase transitions and the variation of transition temperatures with the increase of co-monomer AAm percentage as (a) 5%, (b) 10%, and (c) 15% in total 3 mmol of NIPAAm and AAm monomers. All samples were conducted for five continuous heating–cooling cycles. more AAm content displays a higher LCST than those with less AAm content, which
is contrary to the observation for PNIPAAm polymer shell. These results also suggest
that LCST can be accurately controlled to shift for a few degrees, which is ca. 2.0–2.5
˚C increase per 0.15 mmol AAm addition in the present work. For in vivo applications,
the LCST should be tuned to a value above the body temperature (37 ˚C) but below
the hyperthermia temperature (42 ˚C).215 Our system with subtle LCST tuning
properties indicates the possibility to manipulate the drug release temperature for such
application.
Figure 3.11a shows that Fe3O4@mSiO2@P(NIPAAm-co-AAm) NPs have a very
weak interaction with external magnetic field (300 mT) at room temperature and can
be stable for few hours, which is usually seen for superparamagnetic NPs with
diameter smaller than 20 nm. It is obviously not suitable for magnetic separation
Figure 3.11 Photographs of Fe3O4@mSiO2@P(NIPAAm-co-AAm) aqueous suspension under the influence of a magnetic field (300 mT) at (a) 20 ˚C and (b) 40 ˚C.
34
applications. Currently, large magnetic particles or beads usually with diameters of
few micrometers are applied for magnetic separation because of their rapid and strong
response to external magnetic field. However, their low surface-to-volume ratio and
large hydrodynamic size limit their conjugation efficiency and circulation time, i.e.
blood half life. Figure 3.11b shows that the thermo-sensitive Fe3O4@mSiO2@
P(NIPAAm-co-AAm) NPs can respond to an external magnetic field rapidly when the
temperature is above their LCST because of their hydrophobic surface and forming
aggregation, similar with the previous reported PNIPAAm coated magnetic
microspheres.216 This behavior is reversible over several cycles and the polymer
coated NPs became soluble again when temperature is below LCST.
3.3 High aspect ratio gold nanorods
3.3.1 Morphological and structural studies
The effects of different concentrations of HNO3 (0.2-72 mM) on the morphology
of gold nanorods have been studied. Parts of the TEM micrographs are presented in
Figure 3.12. The length and AR of gold nanorods were found to be greatly influenced
by the HNO3 concentration, and the average length of gold nanorods varied from ~21
to 447 nm and the AR changed from ~1 to 26. The highest AR was achieved using 3
mM HNO3, while a further increase of HNO3 concentration resulted in the formation
of shorter nanorods, and finally spherical NPs were obtained at a concentration of 72
mM HNO3 (Figure 3.12d).
Figure 3.12 TEM images of gold nanorods prepared by using HNO3 with the following concentrations: (a) 0, (b) 1 mM, (c) 3 mM, and (d) 72 mM in the solution.
Functional Materials Division, KTH, 2011 35
Figure 3.13 (a) TEM low resolution image of an individual gold nanorods prepared with 1 mM HNO3 and the SAED pattern (inset); (b) HRTEM images showing lattice fringes at the edge of the gold nanorods; (c) XRD pattern of high AR gold nanorods prepared with 1 mM HNO3 and deposited onto a glass slide.
The crystal structure of gold nanorods was studied by HRTEM on a
representative sample prepared in the presence of 1 mM HNO3 solution. The
micrograph (Figure 3.13a) shows typical faceted ends of nanorods. The selected area
electron diffraction (SAED) pattern of this nanorod (inset; Figure 3.13a) exhibits
additional reflections indicating twinning. An HRTEM image of the end region of the
gold nanorod (Figure 3.13b) shows lattice fringes with a d-spacing of 2.35 Å, which is
assigned to Au (111) planes. Gold nanorods were also examined by XRD (Figure
3.13c) where a very strong (111) diffraction peak was observed, which indicates that
the evaluation from the HRTEM image of a single nanorod is valid for the whole
ensemble.
3.3.2 Particle growth mechanism studies
An attempt to elucidate the mechanism of formation of high AR gold nanorods
was carried out by studying the morphology and crystal structure of gold nanorods
formed at different growth stages through HRTEM. Figure 3.14a (inset) shows images
of gold NPs (AR ~1.2) which were formed 5 min after the introduction of the
reducing agent in the presence of 1 mM HNO3. Most of the formed particles were
found to be twinned, indicated by the crystal lattices pointing to different directions
but with same lattice spacing (Figure 3.14a). These gold NPs grew anisotropically
36
Figure 3.14 TEM images of gold NPs synthesized in the presence of 1 mM HNO3 showing crystal lattice and morphology (inset) at a growth duration of (a) 5 min, (b) 1 h; and (c) Fourier domain image of the particle in (a) by FFT process shows three pairs of spots with the same radial distance (indicated by arrows) that corresponds to the three marked lattice fringes in (a). All scale bars in white are 5 nm. into rod-shaped particles with ARs of ~17 after 1 h (Figure 3.14b). Fast Fourier
transform (FFT) analysis of Figure 3.14a exhibits several pairs of spots (indexed in
the Figure 3.14c) with the same radial distance from the center spot, indicating that
these spots represent the same crystal lattice plane and that their mutual orientation is
due to twinning. Exploration by inverse FFT (see Supplementary material in
corresponded paper) shows that the lattice spacing of these five-fold twinned gold
NPs is of ca. 2.4 Å, close to that of the Au (111) lattice plane.
Further examination of the effect of nitric acid on the growth of gold NPs (Figure
3.15) shows that not only the morphology but also the crystal structure of gold NPs
was greatly influenced by the concentration of HNO3. For the nanorods produced with
72 mM HNO3, only single-crystal spherical NPs were formed at the initial stage,
different from the twinned crystal structure when using 1 mM HNO3. Furthermore,
these NPs grew isotropically, instead of anisotropically, with their diameter increasing
from ~7 to ~15 nm within 1 h (insets of Figure 3.15a-b). Under these conditions,
about 6% (12 out of 200) of the NPs showed five-fold twinned decahedral shape
(Figure 3.15b), while they did not grow into rod-shaped particles. The HRTEM image
of the initially formed NPs (Figure 3.15a) shows only one set of clearly resolved
lattice fringes (indicated by arrows) and the pair of spots (with radial symmetry) in
Fourier domain image (Figure 3.15c) corresponds to that lattice fringes. Inverse FFT
analysis shows that the lattice spacing is ~2.0 Å, which matches with an Au (200)
Functional Materials Division, KTH, 2011 37
Figure 3.15 TEM images of gold NPs synthesized in the presence of 72 mM HNO3 showing crystal lattice and morphology (inset) at a growth durations of (a) 5 min, and (b) an example of few particles with decahedron structures after 1 h; (c) Fourier domain image of the particle in (a) (the one with the lattice plane indicated) by FFT process. The pair of the most intense spots (with radial symmetry), which is indicated by an arrow, corresponds to the most intense lattice fringes in the HRTEM image. All scale bars in white are 5 nm. lattice plane and confirms that these single-crystal gold NPs were grown on {100}
lattice planes (see Supplementary material in corresponded paper).
We hypothesize that the strong oxidative dissolution of Au(0) seeds at high
concentration of HNO3 in the presence of chlorine ions is responsible for no existence
of twin defects at the earlier stage of NP growth, which are essential for the
anisotropic growth of NPs into rods.217 It is noticed that HNO3 has a lower reduction
potential than HAuCl4, so its oxidative property can only be effective at high
concentration. Conversely, multiple-twinned gold NPs were formed intermediately
and grew into elongated nanorods at low HNO3 concentration, which implies that the
effect of nitrate ions on elongation of the surfactant micelles218 is predominant over
the oxidative dissolution of multiple twinned particles, the former effect being
responsible for growth of long gold nanorods. On the other hand, the oxidative
dissolution by HNO3 would assist the molecules on the surface of a small
(energetically unstable) particle to diffuse and then deposit onto unbound (111) facets
of larger particles to lower the overall energy of the system, i.e., Ostwald ripening.219
Therefore, the mechanism of growth of high AR gold nanorods can be attributed to
the effect on elongation of surfactant micelles by nitrate ions and the process of
Ostwald ripening. Consequently, there is only a narrow window of HNO3
concentration that results in high AR gold nanorods.
38
3.3.3 Biocompatibility evaluation of high AR gold nanorod
3.3.3.1 Viability studies
MDDCs cultured in medium alone were used as a negative control, cells treated
with LPS (0.1 µg/mL) as a positive control, while spherical gold NPs were used as a
reference.199 It is found that nanorods with high AR seem not to have an impact on
cell viability (Figure 3.16a, b) and similar results were seen for the spherical gold NPs.
In contrast, nanorods with low AR at a concentration of 50 µg/mL induced a
considerable decrease in viability from 84 ± 6.6 % (medium control) to 57 ± 18 %
(Figure 3.16a), involving mainly late apoptosis (programmed cell death) and necrosis
(uncontrolled cell death) (Figure 3.16b). At the two lower concentrations of similar
type of nanorods, no increased cell death was observed (Figure 3.16a) compared to
the medium control.
To elucidate the different effects seen for the two rod types, the chemicals used
during the two production processes were investigated in the same viability analysis
as above. The amounts of chemicals used during the particle preparations were added
to MDDCs without nanorods, as well as ten fold lower amounts, to simulate the
dilution caused during the particle washing steps. AgNO3 was only used for the low
AR rods and HNO3 for the high AR rods. However, neither of these precursors nor
L-ascorbic acid seems to have any considerable effects on viability (Figure 3.16c, d).
CTAB did so and is concentration dependent.
In a second set of experiments, we used a larger number of washing steps of the
rods to reduce the amount of CTAB bound on gold nanorods. Hereby improved
biocompatibility was achieved for the low AR rods, but it did also cause particle
agglomeration. This suggests that the observed decrease of viability induced by the
low AR rods were not due to different morphology, but rather due to higher
concentrations of CTAB, which could not be efficiently washed away compared to the
case of high AR rods. We propose that the addition of HNO3, used for synthesis of
high AR rods, changes the equilibrium of the chemical reaction in the colloidal
Functional Materials Division, KTH, 2011 39
Figure 3.16 Flow cytometry data showing percentage viable cells (a, c), and percentage early, and late apoptotic and necrotic cells (b, d) after 24 h co-culture with either NPs (a, b) or with chemicals used for the preparations of high (~21) and low AR (~4.5) gold nanorods (c, d) Results represent the mean ± SEM (standard error of the mean) from 3 (a, b) or 4 (c, d) experiments, respectively, using cells from different healthy blood donors. MDDCs cultured in medium alone were included as a negative control and LPS was included as a positive control.
suspension, thereby facilitating the removal of CTAB molecules from the surface of
gold nanorods. The results from our MDDC cultures show that this is important for
their higher biocompatibility.
3.3.3.2 Immune modulatory effects
The biocompatibility of the high AR gold nanorods were further evaluated in
terms of immune modulatory effects on MDDCs, after co-culture for 24 h. Results
showed that the rods did not have any impact on the overall expression of CD40,
CD80, CD83 or CD86 on MDDCs as measured by mean fluorescence intensity
40
Figure 3.17 Expression of the MDDC surface molecules CD40, CD80, CD83 and CD86 were measured by flow cytometry after 24 h co-culture with spherical gold NPs or high AR (~21) gold nanorods and are expressed as mean fluorescence intensity (MFI). For each sample, 104 cells within the gate for viable MDDCs were analyzed. LPS was used as a positive control. Results represent the mean ± SEM from 3 independent experiments using cells from different healthy blood donors.
(Figure 3.17). However, the percentage of CD86+ cells was slightly increased in a
dose dependent manner, as seen in Figure 3.18, which confirms our previous study on
spherical gold NPs.199 We can therefore conclude that neither the high AR nanorods
nor the spherical NPs induced any pronounced maturation of the MDDCs, thus
different morphologies of gold NPs did not seem to cause any different cellular
responses.
Figure 3.18 Expression of the MDDC surface molecules CD40, CD80, CD83 and CD86 were analyzed by flow cytometry after 24 h and are expressed as percentage of positive cells. For each sample, 104 cells within the gate for viable MDDCs were analyzed. LPS was used as a positive control. Results represent the mean ± SEM from 3 independent experiments using cells from different healthy blood donors.
Functional Materials Division, KTH, 2011 41
3.3.3.3 Cellular internalization study
To explore the interaction between high AR rods and MDDCs, we also
investigated whether the gold nanorods are internalized by MDDC using TEM.
Within 1 h of co-incubation with nanorods (50 µg/mL), it could be detected that these
nanorods were inside the cells and packed within vesicular structures, probably
endolysosomes.220 After 24 h most cells contained high amounts of nanorods (Figure
3.19a, b) with a similar uptake in vesicular structures as at 1 h, demonstrating an
efficient uptake by MDDCs. These observations were also seen with the spherical
gold NPs. The relationship between the surface coating of gold nanorods and cellular
uptake has been studied previously, and it was found that the binding of serum
proteins plays a significant role in nanorods uptake.221 However, the MDDC uptake of
the gold nanorods analyzed in this study requires further investigation.
Figure 3.19 TEM images of MDDCs show internalized high AR (~21) gold nanorods after 24 h (a), which reside within vesicular structures, as visualized by increased magnification (b).
42
4 Conclusions
In this thesis, multifunctional nanomaterials have been developed through various
methodologies for biomedical applications, such as contrast agents for MRI and
thermo-sensitive magnetic separation.
The mSiO2 is coated on a single core of Fe3O4 NP to form a uniform core-shell
structure with tunable coating thickness. We have investigated parameters responsible
for producing high quality core-shell structures. Relaxivity studies of these core-shell
NPs showed better contrast properties with enhanced r2/r1 ratio as a function of the
coating thickness, which is 20 fold higher than that of the commercial contrast agents.
We ascribe the mechanism of such enhancement to the effect of mSiO2 coating layers
on water exclusion and diffusion.
A multifunctional nanomaterial has been developed by grafting a
thermo-sensitive copolymer P(NIPAAm-co-AAm) as the outer shell onto
Fe3O4-mSiO2 NPs. In addition to the superparamagnetic property, high r2 relaxivity,
and large surface area, we found that the phase transition temperature of the core-shell
composite can be finely adjusted from ca. 34 to ca. 42 ˚C by tuning the quantity or
composition of the co-monomers. The integrated capabilities from different
components of the multifunctional NPs make these materials a novel candidate for
future medical diagnosis and therapy.
A new synthesis method of gold nanorods with high AR has been developed
without using pre-made seeds of gold NPs. The growth mechanism of high AR gold
nanorods has been explained by the effects of HNO3 on elongation of micelles and
oxidative dissolution of Au(0) together with Ostwald ripening. The biocompatibility
of the nanomaterials is evaluated by studying the immune modulatory effects with
MDDCs. High AR gold nanorods are found no impact on the viability of primary
MDDCs and no significant effect on their cell surface molecules involved in T cell
activation, even though the particles were efficiently internalized by the cells, which
exhibit a high potential of this material for medical applications.
Functional Materials Division, KTH, 2011 43
Future work
A few designs for construction of multifunctional NPs can be developed for
several biomedical applications, namely thermo-therapy, dual-modality MRI, and
drug release.
Specifically, one possible hierarchical structure may be composed by growing Au
nanorods in the mesoporous channels of Fe3O4@mSiO2 NPs to enable both magnetic
imaging and NIR-induced local heating simultaneously for the improvement of
medical diagnosis and therapy. Certain surface functionalization of mSiO2 will
facilitate the realization of other applications, such as cancer cell targeting by
conjugating with folic acid through aminopropyltriethoxysilane (APTMS), magnetic
separation or targeting using thermo-sensitive polymer.
A proper design on construction of assembly of different magnetic materials
could be useful for dual modal MRI, where two different T1 and T2 imaging modes are
achieved simultaneously, potentially providing highly accurate information. For such
purpose, typical T1 contrast agents Gd-chelate or amorphous Gd2O(CO3)2 can be
attached or formed in situ on the surface of Fe3O4@mSiO2. This system may be
potentially capable for the imaging of a wide range of biological targets with
enhanced diagnostic accuracy.
For controlled drug release, one possible design incorporates the synthesis of a
copolymer of thermal sensitive polymers (e.g. PNIPAAm) and biodegradable
polymers (e.g. PLLA, PDLA, PLGA) as drug carriers. Their hydrophobic interior can
be loaded with water-insoluble anticancer drug and photosensitive agent (e.g. initially
hydrophilic Au nanorods phase transferred to non-polar solvent) for thermo-triggered
drug release.
44
Acknowledgements
First of all, I would like to thank my supervisor, Prof. Mamoun Muhammed, for
giving me the precious opportunity to join the Functional Materials Division and
leading me to the world of nanotechnology. His enlightening ideas and kindness are
always strong supports during my study.
I would like to thank Assoc. Prof. Muhammet Toprak for his great help from
research to everyday life, especially for his kind help in revising my manuscripts. I
wish him a great success in the scientific career.
I want to thank Dr. Jian Qin for his great patience, scientific discussion and
professional guidance on my first step of research. Thanks to Dr. Shanghua Li and Dr.
Andrea Fornara for the help, encouragement, and invaluable advices. I wish all of
them a splendid future for their career either in ABB or YKI, as well for the family. I
am very grateful to Abhi for the thorough linguistic check of the thesis. Many thanks
to the members in Functional Materials Division, Dr. Salam, Ying, Xiaodi, Sverker,
Carmen, Robina, Mazher, Terrance, Mohsin, Nader, Yichen, Dr. Mohammed and all
former colleagues, for their friendship and support. I appreciate Hans and Wubshet for
their great work of maintaining and helping on use of electron microscopes.
I am grateful to my coworkers from Karolinska Institute for the fruitful
collaborations: Dr. Helen Vallhov, Dr. Britta Andersson, Assoc. Prof. Susanne
Gabrielsson and Prof. Annika Scheynius from Department of Medicine; Neus Feliu
and Prof. Bengt Fadeel from Institute of Environmental Medicine. I would also like to
thank Dr. Sophie Laurent, Dr. Alain Roch and Prof. Robert N. Muller from University
of Mons in Belgium for the productive collaboration and great help on MR
experiments. Many thanks to Lin Dong and Assoc. Prof. Sergei Popov from Optics
Division, KTH, for the great collaboration.
Last but not least, I would like to thank my parents for their endless support and
encouragement, and the most important person, my wife Min Tian, for her deep love
and a strong support for my scientific career.
Functional Materials Division, KTH, 2011 45
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