Fei Ye PhD529974/FULLTEXT01.pdf · 2012. 5. 31. · iv 6G/gold nanoparticles: impact of SiO2...

Chemically Synthesized Nano-Structured Materials for

Biomedical and Photonic Applications

Fei Ye

叶飞

Doctoral Thesis

Stockholm 2012

Functional Materials Division

School of Information and Communication Technology

Royal Institute of Technology (KTH)

Postal address Functional Materials Division

School of Information and

Communication Technology

Royal Institute of Technology

Electrum 229

Isafjordsgatan 22

SE 164 40, Kista/Stockholm, Sweden

Supervisor Prof. Mamoun Muhammed

Email: [email protected]

Co-supervisor Assoc. Prof. Muhammet S. Toprak

Email: [email protected]

TRITA-ICT/MAP AVH Report 2012:12

ISSN 1653-7610

ISRN KTH/ICT-MAP/AVH-2012:12-SE

ISBN 978-91-7501-416-6

© Fei Ye, 2012

Universitetsservice US AB, Stockholm 2012

Functional Materials Division, KTH, 2012 i

Abstract

Nanostructured materials have attracted a broad interest for applications in scientific and engineering fields due to their extraordinary properties stemming from the nanoscale dimensions. This dissertation presents the development of nanomaterials used for different applications, namely biomedicine and dye lasing.

Various inorganic nanoparticles have been developed as contrast agents for non-invasive medical imaging, such as magnetic resonance imaging (MRI) and X-ray computed tomography (CT), owing to their unique properties for efficient contrasting effect. Superparamagnetic iron oxide nanoparticles (SPIONs) are synthesized by thermo-decomposition method and phase-transferred to be hydrophilic used as MRI T2 (negative) contrast agents. Effects of surface modification of SPIONs by mesoporous silica (mSiO2) coating have been examined on the magnetic relaxivities. These contrast agents (Fe3O4@mSiO2) were found to have a coating-thickness dependent relaxation behavior and exhibit much higher contrast efficiency than that for the commercial ones. By growing thermo-sensitive poly(N-isopropylacrylamide -co-acrylamide) (P(NIPAAm-co-AAm)) as the outermost layer on Fe3O4@mSiO2 through free radical polymerization, a multifunctional core-shell nano-composite has been built up. Responding to the temperature change, these particles demonstrate phase transition behavior and were used for thermo-triggered magnetic separation. Their lower critical solution temperature (LCST) can be subtly tuned from ca. 34 to ca. 42 ˚C, suitable for further in vivo applications. An all-in-one contrast agent for MRI, CT and fluorescence imaging has been synthesized by depositing gadolinium oxide carbonate hydrate [Gd2O(CO3)2·H2O] shell on mSiO2-coated gold nanorod (Au NR), and then the particles were grafted with antibiofouling copolymer which can further link with the fluorescent dye. It shows both a higher CT and MRI contrast than the clinical iodine and gadolinium chelate contrast agent, respectively. Apart from the imaging application, owing to the morphology of Au NR, the particle has a plasmonic property of absorbing near-infrared (NIR) irradiation and suitable for future photothermal therapy. Cytotoxicity and biocompatibility of aforementioned nanoparticles have been evaluated and minor negative effects were found, which support their further development for medical applications.

Gold nanoparticles embedded in the optical gain material, water solution of Rhodamine 6G (Rh6G) in particular, used in dye lasers can both increase and damp the dye fluorescence, thus, changing the laser output intensity. The studies of size effect and coating of gold nanoparticles on photostability of the gain media reveal that small sized (ca. 5.5 nm) gold nanoparticles are found detrimental to the photostability, while for the larger ones (ca. 25 nm) fluorescence enhancement rather than quenching is likely to occur. And a noticeable improvement of the photostability for the gain material is achieved when gold is coated with SiO2.

TRITA-ICT/MAP AVH Report 2012:12, ISSN 1653-7610

ISRN KTH/ICT-MAP/AVH-2012:12-SE, ISBN 978-91-7501-416-6

Functional Materials Division, KTH, 2012 iii

LIST OF PAPERS

This thesis is based on the following papers, which are referred to in the text by Roman numerals. All previously published papers were reproduced with permission from the respective publishers.

I. 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”, International Journal of Nanotechnology, 2011, 8, 631-652

II. Fei Ye, Jian Qin, Muhammet S. Toprak and Mamoun Muhammed,

“Multifunctional core-shell nanoparticles: superparamagnetic, mesoporous, and thermo-sensitive”, Journal of Nanoparticle Research, 2011, 13, 6157-6167

III. Fei Ye, Sophie Laurent, Andrea Fornara, Laura Astolfi, Jian Qin, Alain Roch,

Alessandro Martini, Muhammet S. Toprak, Robert N. Muller and Mamoun Muhammed, “Uniform mesoporous silica coated iron oxide nanoparticles as a highly efficient, nontoxic MRI T2 contrast agent with tunable proton relaxivities”, Contrast Media and Molecular Imaging, 2012, in press, DOI: 10.1002/cmmi.1473

IV. Fei Ye, Torkel Brismar, Jingwen Shi, Lin Dong, Ramy El Sayed, Sergei Popov,

Muhammet S. Toprak and Mamoun Muhammed, “Gold nanorod/mesoporous silica/gadolinium oxide carbonate hydrate core/shell nanoparticles: A multimodal contrast agent for MRI, CT and fluorescence imaging”, 2012, Manuscript

V. 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”, Toxicology and Applied Pharmacology, 2011, 253, 81-93

VI. 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, 2010, 1257, 1257-O04-03

VII. 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-radiativedecay”, Journal of the European Optical Society−Rapid Publications, 2011, 6, 11019

VIII. Lin Dong, Fei Ye, Adnan Chughtai, Sergei Popov, Ari T. Friberg and Mamoun

Muhammed, “Photostability of lasing process from water solution of Rhodamine 6G with gold nanoparticles”, Optics Letters, 2012, 37, 34–36; also in Virtual Journal for Biomedical Optics, 2012, Vol. 7, Iss. 3.

IX. Lin Dong*, Fei Ye*, Adnan Chughtai, Vytautas Liuolia, Sergei Popov, Ari T.

Friberg and Mamoun Muhammed, “Lasing from water solution of Rhodamine

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6G/gold nanoparticles: impact of SiO2 coating on metal surface”, Submitted to IEEE Journal of Quantum Electronics, 2012 (*Shared first authorship)

Other work not included:

I. Jiantong Li, Fei Ye, Sam Vaziri, Mamoun Muhammed, Max C. Lemme and Mikael Östling, “A simple route towards high-concentration surfactant-free graphene dispersions”, Carbon, 2012, 50, 3113-3116

II. Jingwen Shi, Ylva Rhode, Clara Ersson, Sylvain Geny, Fei Ye, Mamoun Muhammed, Edvard Smith and Lennart Möller, “Amino-modified silica nanoparticles as non-viral vectors for the delivery of plasmid DNA”, 2012, Manuscript

Functional Materials Division, KTH, 2012 v

Conference presentations 1. Lin Dong, Fei Ye, Adnan Chughtai, Sergei Popov, Ari T. Friberg and Mamoun

Muhammed, “Enhanced photostability of aqueous solution of Rhodamine 6G with gold nanoparticles in lasing process by silica coating” (oral), CLEO:2012, May 6-11, 2012, San Jose, CA, USA

2. Lin Dong, Fei Ye, Sergei Popov, Ari T. Friberg and Mamoun Muhammed, “Fluorescence enhancement of Rhodamine 6G by SiO2-coated gold nanoparticles” (oral), 2011 Asia Communications and Photonics Conference, Nov 13-16, 2011, Shanghai. CHINA

3. Fei Ye, Jian Qin, Muhammet S. Toprak and Mamoun Muhammed,

“Multifunctional core-shell nanoparticles: superparamagnetic, mesoporous, and thermo-sensitive” (poster), 10th International Conference on Nanostructured Materials, Sept 13-17, 2010, Rome, ITALY

4. Daniel Bacinello, Andrea Fornara, Jian Qin, Fei Ye, Muhammet Toprak and

Mamoun Muhammed, “Laser triggered drug release from smart polymeric nanospheres containing gold nanorods” (poster), 10th International Conference on Nanostructured Materials, Sept 13-17, 2010, Rome, ITALY

5. 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” (poster), 12th Bi-Annual Conference on Contrast Agents and Multimodal Molecular Imaging, May 19-21, 2010, Mons, BELGIUM

6. 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” (oral), Materials Research Society Spring meeting (2010 MRS), Apr 5-9, 2010, San Francisco/California, USA

7. 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”(oral), 34th International Conference and Exposition on Advanced Ceramics and Composites, Jan 24-29, 2010, Daytona Beach, Florida, USA

8. 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” (oral), 2009 Asia Communications and Photonics Conference and Exhibition, Nov 2-6, 2009, Shanghai. CHINA

vi

9. 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” (poster), EuroNanoMedicine 2009, Sept 28-30, 2009, Bled, SLOVENIA

10. 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, Aachen, Germany, 2008) Vol. 2: Materials Science, pp. 209-210, DOI: 10.1007/978-3-540-85226-1_105

11. Fei Ye, Jian Qin and Mamoun Muhammed, “Cellular uptake and cytotoxicity of

gold nanorods” (oral), Nanoimmune Workshop, Feb 13, 2007, Uppsala, SWEDEN

Functional Materials Division, KTH, 2012 vii

Contributions of the author

Paper I. Participated in initiating the idea, planning of experiments, coordinating collaboration, preparing the materials, performing material characterization, evaluation of the results, and writing main parts of the article. Paper II. Initiating the idea, planning of experiment, preparing the materials, performing material characterization, evaluation of the results, and writing the article. Paper III. Initiating the idea, planning of experiment, coordinating collaboration, preparing the materials, performing material characterization, evaluation of the results, and writing main parts of the article. Paper IV. Initiating the idea, planning of experiment, coordinating collaboration, preparing the materials, performing material characterization, evaluation of the results, and writing main parts of the manuscript. Paper V. Preparing some of the materials, performing morphological characterization, and writing parts of the article. Paper VI. Preparing some of the materials, performing morphological characterization, and writing parts of the article. Paper VII. Participated in initiating the idea, preparing the materials, performing material characterization, and writing part of the article. Paper VIII. Participated in initiating the idea, preparing the materials, performing material characterization, and writing part of the article. Paper IX. Participated in initiating the idea, preparing the materials, performing material characterization, and writing part of the article.

Functional Materials Division, KTH, 2012 ix

ABBREVIATIONS AND SYMBOLS

AAc acrylic acid AAm acrylamide AAS atomic absorption spectroscopy AFM atomic force microscopy AIBN 2,2'-azobisisobutyronitrile AR aspect ratio ATRP atom transfer radical polymerization BET Brunauer-Emmett-Teller (method for surface area analysis) CT computed tomography CTAB cetyltrimethylammonium bromide DLS dynamic light scattering DMEM Dulbecco’s modified Eagle medium DMF N,N-dimethylformamide DMSO dimethylsulfoxide DSC differential scanning calorimetry EDS energy-dispersive X-ray spectroscopy EDTA ethylenediaminetetraacetic acid EtOAc ethyl acetate FBS fetal bovine serum FDA Food and Drug Administration FFT fast Fourier transformation FITC fluorescein isothiocyanate FTIR Fourier transform infrared HRTEM high resolution transmission electron microscopy ICP-AES inductively coupled plasma-atomic emission spectrometry KPS potassium persulfate LCST lower critical solution temperature, [°C] LPS lipopolysaccharide LSPR localized surface plasmon resonance M magnetization, [emu g-1] or [A m2 kg-1] MBA N,N’-methylene bisacrylamide MCM Mobil crystalline materials MDDC monocyte-derived dendritic cells MR magnetic resonance MRI magnetic resonance imaging MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-

2-(4-sulfophenyl)-2H-tetrazolium, inner salt MTT 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide MUDA 11-mercaptoundecanoic acid Nd:YAG neodymium-doped yttrium aluminium garnet; Nd:Y3Al5O12 NIPAAm N-isopropylacrylamide

x

NIR near-infrared NLDFT nonlocal density functional theory NMR nuclear magnetic resonance NMRD nuclear magnetic resonance dispersion NP nanoparticle NR nanorod OA oleic acid OLA oleylamine PBS phosphate-buffered saline PCS photon correlation spectroscopy PDLA poly(D,D-lactide) PEG poly(ethylene glycol) PEGMA poly(ethylene glycol) methyl ether methacrylate 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 SAED selected area electron diffraction SEM scanning electron microscope 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 TGA thermogravimetric analysis THF tetrahydrofuran TMSMA 3-(trimethoxylsilyl) propyl methacrylate TOAB tetraoctylammonium bromide UV-Vis ultraviolet-visible UV-Vis-NIR ultraviolet-visible-near infrared 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, 2012 xi

Table of contents

ABSTRACT…………………………………………………………………………...I LIST OF PAPERS…………………………………………………………………..III ABBREVIATIONS AND SYMBOLS………………………………...…………...IX TABLE OF CONTENTS…………………………………………………………...XI 1 INTRODUCTION ................................................................................................ 1

1.1 OBJECTIVES .............................................................................................................. 1 1.2 OUTLINE .................................................................................................................... 1 1.3 MULTIFUNCTIONAL NANOMATERIALS ............................................................. 3

1.3.1 Hierarchical nanostructures ................................................................................. 3 1.3.2 Functional nanomaterials for biomedical applications ........................................ 4

1.4 BIOMEDICAL APPLICATIONS OF NANOMATERIALS ....................................... 6 1.4.1 Visualization .......................................................................................................... 6

1.4.1.1 Magnetic resonance imaging (MRI) ........................................................... 6 1.4.1.2 X-ray computed tomography (CT) ............................................................. 8 1.4.1.3 Fluorescence imaging ................................................................................. 9

1.4.2 Thermo-sensitive drug delivery ............................................................................. 9 1.4.3 Plasmonic photothermal therapy ........................................................................ 10

1.5 PREPARATION METHODS FOR NANOPARTICLES ........................................... 10 1.6 GOLD NANOPARTICLES FOR LASING APPLICATION ..................................... 11

2 EXPERIMENTAL METHODOLOGY…...…………………………………..13 2.1 SYNTHESIS OF INORGANIC NANOPARTICLES ................................................ 13

2.1.1 Superparamagnetic iron oxide nanoparticles (SPIONs) ..................................... 13 2.1.2 Mesoporous silica ............................................................................................... 13 2.1.3 Gadolinium oxide carbonate hydrate and gadolinium oxide .............................. 13 2.1.4 Au nanocrystals with different morphologies ...................................................... 13

2.2 SURFACE MODIFICATION AND FUNCTIONALIZATION ................................. 14 2.2.1 Coating Fe3O4 and Au with amorphorous (mesoporous) silica .......................... 14 2.2.2 Coating Gd2O(CO3)2·H2O on mesoporous silica……………………………... ...... 15 2.2.3 Antibiofouling copolymer coating on Gd2O(CO3)2·H2O ..................................... 15 2.2.4 Grafting thermo-sensitive copolymer from surface of mesoporous silica ........... 16

2.3 GOLD NANOPARTICLES IN LASER GAIN MEDIA ............................................ 16 2.4 CHARACTERIZATIONS .......................................................................................... 16 2.5 BIOCOMPATIBILITY EVALUATION ..................................................................... 18

2.5.1 Cell/cell lines and culture conditions .................................................................. 18 2.5.2 Cell viability assay .............................................................................................. 18

3 RESULTS AND DISSCUSSIONS ..................................................................... 20 3.1 SUPERPARAMAGNETIC MRI CONTRAST AGENTS ......................................... 20

3.1.1 Magentic mesoporous nanoparticles ................................................................... 20 3.1.2 Magnetic and MR studies of Fe3O4@mSiO2 nanoparticles ................................ 21 3.1.3 Relaxometric properties ...................................................................................... 22 3.1.4 Cell viability assessment ..................................................................................... 23

3.2 MULTIMODAL MRI, CT, AND FLUORESCENCE AGENTS ............................... 23 3.2.1 Morphological, structural, and elemental analyses ............................................ 23 3.2.2 MRI, CT, and fluorescence imaging .................................................................... 24 3.2.3 Biocompatibility evaluation ................................................................................ 25

3.3 MULTIFUNCTIONAL CORE-SHELL COMPOSITE NANOPARTICLES ............ 26 3.3.1 Morphology, structure, and magnetic properties ................................................ 26 3.3.2 Thermo-responsive properties and manipulation of LCST ................................. 26

3.4 HIGH ASPECT RATIO GOLD NANORODS .......................................................... 27 3.4.1 Morphological and structural studies ................................................................. 27 3.4.2 Particle growth mechanism studies ..................................................................... 28

xii

3.4.3 Biocompatibility evaluation ................................................................................ 29 3.4.3.1 Viability studies ........................................................................................ 29 3.4.3.2 Immune modulatory effects...................................................................... 29 3.4.3.3 Cellular internalization study ................................................................... 30

3.5 EFFECTS OF GOLD NANOPARTICLES ON LASING PERFORMANCE ........... 30 3.5.1 Gold nanoparticles and Au@SiO2 ...................................................................... 30 3.5.2 Influence of particle concentration and size ....................................................... 32 3.5.3 Influence of surface coating ................................................................................ 33

4 CONCLUSIONS………………………………………………………………..34 FUTURE WORK ... …………………………………………………………………36 ACKNOWLEDGEMENTS ………………………………………………………...37 REFERENCES ... ……………………………………………………………………38

Functional Materials Division, KTH, 2012 1

1 Introduction Nanotechnology is a rapidly progressing field, which have a tremendous impact

on interdisciplinary areas of research such as materials, electronics, and medicine.

Nanomaterials, at least one dimension sized in nanoscale, are the key components for

development of nanotechnology due to their extraordinary physical, chemical, and

biological properties.1 Due to their comparable size with biological molecules and

specific properties, nanomaterials have been integrated into biomedicine and

numerous applications are realized, such as more efficient diagnostics, enhanced

visualization, targeted drug delivery, and controlled therapeutics, etc.2 Nanomaterials

have also demonstrate tremendous potential in improving the properties of optical

materials and electronics.3, 4

1.1 Objectives The aim of this thesis is to tailor the nanomaterials for multifunctional medical

imaging and stimuli-sensitive applications. The effects of gold NPs on laser

performance are also to be investigated.

1). Mesoporous silica (mSiO2) is coated on SPIONs in order to study the

dependence of magnetic relaxivities and contrast enhancement on permeability

property of the coating layers. To incorporate environmental responsive property for

multifunctionality, thermo-sensitive copolymer is grafted from the surface MRI T2

contrast agents. For simultaneous detection with multiple imaging techniques, MRI

contrast agent can be coated outside of a CT contrast agent and further conjugation

with PEG and fluorophore is possible. The impact of different morphologies of gold

nanomaterials on biocompatibility is also investigated.

2). To study the effects of gold NPs in gain media for laser performance,

different sized gold NPs are employed to examine the fluorescence quenching and

photostability of dye. To minimize the photostability deterioration, a layer of charge

insulating material, e.g. SiO2, is going to be coated on gold.

1.2 Outline Parts of this thesis are presented in the previously defended Licentiate Thesis

2

(ISBN: 978-91-7415-903-5).5

Chapter 1.3 briefly introduces strategies for hierarchical structures built-up by

different nanomaterials to simultaneously realize multifunctions. Nanoparticles with

specific functions for biomedicine, e.g. visualization, environmental-sensitive, loading,

and photothermal heating properties, are also introduced. Chapter 1.4 briefly presents

the physical background of some biomedical applications and the use of the functional

nanomaterials. Chapter 1.5 summarizes the chemical methods for synthesis of

various nanomaterials. Chapter 1.6 introduces the research of noble metal

nanomaterials on performance of fluorophores and optical applications.

Chapter 2.1 begins with the description of preparation of various inorganic

nanomaterials. In Chapter 2.2, surface modification and functionalization of different

materials are summarized. Preparation of gold NPs and organic dye for fluorescence

and lasing measurement is described in Chapter 2.3. Following characterization

methods by different techniques in Chapter 2.4, biocompatibility evaluation of

different materials is reported in Chapter 2.5.

Chapter 3 discusses the results of synthesis and properties of nanomaterials for a

variety of applications. In Chapter 3.1, Fe3O4@mSiO2 core-shell NPs with variable

coating thicknesses are discussed on their efficiency for MRI T2 contrast enhancement.

The impacts of mSiO2 coating thickness on the magnetic relaxivities are evaluated.

Chapter 3.2 demonstrates a hierarchical structured nanostructure with combination of

several functional materials for MRI, CT, and fluorescence imaging with enhanced

contrast compared with the clinical ones. In Chapter 3.3, properties of thermo-

sensitive polymer PNIPAAm coated Fe3O4@mSiO2 NPs are studied, and the thermal

behaviors of these nanocomposite is manipulated by adjusting the composition of the

monomers. Chapter 3.4 discusses the effects of nitric acid on the formation of high

AR gold NRs. The biocompatibility of high AR gold nanorods was evaluated by

testing their viability and immune modulatory effects on human primary MDDCs, and

compared with the effects caused by low AR gold NRs and spherical gold NPs.

Chapter 3.5 discusses the impacts of size and surface coating of gold NPs in gain

media on the fluorescence enhancement and photostability during lasing process.


1.3 Multifunctional nanomaterials

1.3.1 Hierarchical nanostructures

Multifunctional nanomaterials are with high research interest due to the combined

functionalities for simultaneously achieving multiple tasks, which is advantageous

compared with a single functional component. Numerous studies have been conducted

on the development of NPs for imaging,6-12 drug delivery,11-14 biosensing,15, 16 cancer

therapy,7-10 and magnetic cell separation.17 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, to respond to external stimuli, such as

changes of temperature,18 pH value,19 photoirradiation,20 ultrasound,21 biomolecules,22

and magnetic field,23 the sensitive moieties need to be 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.24, 25 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.26 Biocompatibility is another key factor for

medical applications, which means high antigenic compatibility and low toxicity.27

Core-shell structure is one of the most common types of assembly of NPs

bringing together different functional materials into one entity. The core normally is a

solid NP of various materials such as metal,28-30 oxides,11, 31-33 or polymers.34-36

Alternatively, the core can be a sacrificial template for grafting the shell materials, and

then are removed by dissolution,37 etching,38-40 or decomposition41-42 methods. Later,

the payload might be loaded into the interior cavities. Instead of solid cores,

nanostructures with soft cores of oil beads or hydrophobic domain of polymers43, 44

can be prepared by microemulsion method. Liposomes45, 46 and micelles47-50 with

core-shell structures are also functional materials used for therapeutic applications.

Another strategy is to form porous structure for integration of different materials.

4

Porous layers can be grown in situ around the NP cores along their surface-capping

agents,11, 51 or formed by subsequent etching of the condensed shell materials.52 In

another way, porous materials are template-synthesized utilizing surfactants,53 block

copolymers,54, 55 or salts56 as sacrificial pore templates, and other materials can be

grown or loaded in the pores. Very recently, non-toxic porous metal–organic

frameworks have been developed for biomedical applications.57

Assembly of different materials can be also achieved through intimate contact of

two different functional NPs, namely heterodimers or dumbbell-like NPs.58 Other

methods to assemble different components together, instead of direct matching the

crystal planes in dimer NPs, include immobilizing the second component with the

substrate particles through chemical linkage to form core-satellite morphology.59-61

1.3.2 Functional nanomaterials for biomedical applications

Depending on the required function for applications, materials can be categorized

into different species, e.g., drug carriers, imaging probes, thermo-therapy agents, or

stimuli-sensitive moieties.

An ideal drug carrier should be able to deliver the drug to the diseased site,62

possibly through necessary targeting,63, 64 and should not induce any immune response

and should be degradable and produce nontoxic degradation products. Furthermore,

PEGylation of the drug carriers can inhibit the uptake by the RES and increases the

blood circulation time.65 One type of drug carrier is macromolecular conjugates where

the drugs including proteins and antibodies are chemically linked to polymers.

Another type is particulate carriers that entrap the drug in a loading space, providing a

high degree of protection from enzymatic inactivation and can be loaded with various

drugs.66 The most intensively investigated particulate drug carriers are liposomes and

polymeric NPs. A few of liposomal formulations have been approved by FDA for

clinical applications.67, 68 Hydrogels can protect the drug from hostile environments

and also control drug release by changing the gel structures in response to

environmental stimuli.69 Mesoporous materials are also used as drug carriers with the

features of high surface area/pore volume and ordered pore network.70-72


Molecular imaging has emerged recently with diagnostic ability at the molecular

or cellular level.73, 74 Representative non-invasive imaging techniques include optical

imaging, CT, MRI, PET, SPECT, and ultrasound.75 Efforts have been devoted to

develop the contrast agent that can improve the sensitivity and detectability of the

current imaging tools for diagnosing disease76 and tracking therapeutic response.77

Besides the available imaging probes and contrast agents of organic dye11 or

radioactive agents,77 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.78 For instance, QDs show size-tunable absorption

and emission properties, and high fluorescence quantum yields compared to organic

dyes.79 SPION80, 81 are relatively benign contrast agents for MRI with good sensitivity

and low toxicity. Metal-doped ferrite NPs, e.g. MnFe2O4,82 can induce significant MR

contrast-enhancement effects.83 Some other magnetic NPs, such as metal alloys of

FeCo and FePt, Gd2O3,84 and MnO,85 are successfully utilized for in vitro cell labeling

and in vivo T1-weighted MRI.86, 87 Metallic gold NP, NR, nanoshell, and nanocage, are

efficient contrast agents in optical imaging based on their unique SPR properties.88

Inorganic NPs, e.g., magnetic or noble metal particles, 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 field89,

90 or from photo-irradiation.91 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.92, 93 Photothermal therapy (PTT), in which

photothermal agents are employed to achieve the localized heating, is superior to

photodynamic therapy (PDT), where the photosensitizing drug stays in patient body

and is highly sensitive to light. Noble metal NPs94-97 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.

Environmental sensitive materials are of emerging research interest owing to their

potential in many biomedical and technical applications. For instance, significant

6

efforts have been devoted to the development of “smart” delivery systems where

delivery of molecules can be controlled by a variety of external stimuli, e.g.

temperature,98, 99 pH value,43, 100 magnetic field,13 photoirradiation,20 ultrasound,21 and

biomolecules.22 Among the environmentally sensitive nanomaterials, temperature-

responsive materials, e.g., PNIPAAm, are probably one of the most commonly studied

classes,101 and the idea naturally came from the fact that many pathological areas

demonstrate distinct hyperthermia.

1.4 Biomedical applications of nanomaterials

1.4.1 Visualization

1.4.1.1 Magnetic resonance imaging (MRI)

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 protons). 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 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)

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.

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 Gd3+ or Mn2+ complexes, while T2 contrast agents are based on

SPIONs. New generation of nano-particulate contrast agents have being developed to

improve MR contrast with a less amount of dosage.

The magnitude of r1 is determined by accessibility of protons bearing nuclear

spins to the surface of magnetic species,102 and for T1 contrast agent, the lower of r2/r1

8

the better performance for T1 imaging. 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.103 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.104 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 is known as “superparamagnetism”.105 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.

1.4.1.2 X-ray computed tomography (CT)

The use of CT in nuclear medicine imaging has been growing, which could be

combined with PET or SPECT to provide more advanced information, e.g., 3D.106 The

CT machine measures the X-ray transmission through the subject by the detectors and

the calculated values of X-ray attenuation are then represented as gray levels in a 2D

image of the slice. Different tissues have different X-ray absorption and therefore


generate the contrast. Because of the inherent high-contrast resolution of CT,

differences between tissues that differ in physical density by less than 1 % can be

distinguished and the CT value is expressed in Hounsfield units (HU). Clinically,

radiocontrast agents are typically iodine or barium compounds used for detection with

increasing accuracy. Numerous studies have been conducted using more radiodense

(X-ray attenuation) materials, e.g., Au107, 108 and Gd109, instead of iodinated contrast

material for CT measurement. It was reported that contrast agents made from Au NPs

exhibited better contrast properties than commercial ones.110

1.4.1.3 Fluorescence imaging

Fluorescence imaging techniques, e.g., confocal or two-photon microscopy111, are

widely used to provide spatial information by detecting the fluorescence in desired

regions in/on (living) cells. Biological samples are usually stained with or uptake

various fluorophores, e.g., organic dye,11 QDs,112 lanthanide-doped upconversion

nanocrystals,113 and excited by the light and emit fluorescence signals. Conventional

organic dyes or protein fluorophores commonly require specific wavelength for

excitation and have broad emission spectra, as well as affected by fast photobleaching

and subsequent a short fluorescence lifetime.114 While, QDs are semiconductor

nanocrystals with excitons confined in its spatial dimensions, which have broad

excitation spectra, narrow and tunable emission spectra. They are extremely

photo-chemically stable, even after repeated excitation and fluorescence emission,

therefore exhibit high value for imaging applications. Upconversion is the generation

of higher energy light (NIR or IR) from lower energy radiation, using transition metal,

lanthanide, or actinide ions doped into a solid-state host.115 Compared with organic

dyes and semiconductor QDs that require expensive high energy pulse lasers, the

lanthanide-doped materials only need inexpensive NIR diode lasers for excitation due

to the relative high efficiency of the upconversion process. The realization of efficient

NIR to visible upconversion opens a new window for biolabeling and imaging.

1.4.2 Thermo-sensitive drug delivery

For a better therapeutic effect of drug delivery, “on/off” function are important for

10

precise, on-demand drug release. The controlled release of payload can be realized by

various external stimuli. One of the most intensively studies strategies is using

thermo-sensitive polymers together with appropriate drug carriers for controlled drug

release triggered by illuminating with light at resonant wavelengths to enable

photothermal conversion116 or receiving thermal energy from the environment.39

Thermo-sensitive polymers, e.g., family of PNIPAAm,117, 118 undergo a

coil-to-globule transition when temperature is raised from below LCST to above

LCST in aqueous solution. The changes in free energy for both hydrogen bonding and

hydrophobic effect are found responsible for the transition.119, 120 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.

1.4.3 Plasmonic photothermal therapy

Photothermal ablation therapy based on noble metal NPs has been explored for

destroying cancer by employing the heat generated from light for thermal denaturing

of proteins and DNA in the cells, and consequently irreversible damage.95, 121 Desired

noble metal nanostructures for photothermal ablation should have strong and tunable

SPR, high efficiency of photothermal conversion, low toxicity, ease of delivery, and

convenience for bioconjugation for actively targeting cancer cells.

Surface plasma 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 visible till IR region for air/metal and water/metal interfaces.122 The

maximum and bandwidth of surface plasmon band are also influenced by the particle

shape, medium dielectric constant, and temperature.123

1.5 Preparation methods for nanoparticles Preparation of gold nanomaterials can be traced back to Michael Faraday, who

was the first to report the formation of deep-red solutions of colloidal gold, which

later came to be called metallic NPs.124 Later, after the breakthrough of two classical


works as citrate reduction method by Turkevitch125 and two-phase method by Brust,126

nowadays various methods were developed for synthesis of Au NPs, namely

microemulsion,127 seeding growth,128 thermolysis,129 photochemical reduction,130

electrochemical,131 and template132 methods.

Numerous chemical methods have been developed to synthesize magnetic NPs

for biomedical applications. Co-precipitation 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.133 This

method is also applied for synthesis of spinel-type ferromagnets, such as MnFe2O4,134

ZnFe2O4,135 and CoFe2O4.136 Thermo-decomposition method has been developed as

an effect way to synthesize high-quality semiconductor and oxide NPs with controlled

size and shape,137, 138 in which organometallic compounds as precursors are

decomposed in high-boiling non-aqueous media containing stabilizing surfactants.139,

140 Other methods, including microemulsion,141 hydrothermal,142 and sol-gel,143 are

also investigated for synthesis of magnetic NPs. Several phase transfer strategies31, 144,

145 have been applied to hydrophobic magnetic NPs for obtaining their aqueous

dispersion necessary for biomedical applications.

Thermo-sensitive PNIPAAm and its copolymers were synthesized by the radical

polymerization of NIPAAm via a variety of techniques including the typical free

radical initiation in organic solutions146 and redox initiation in aqueous media.147

Other methods include ionic initiators,148, 149 radiation polymerization,150, 151 and the

later investigated living polymerization methods using free radical chemistry, e.g.

RAFT98 and ATRP.152 The polymers obtained by living polymerization methods have

controlled molecular weight and narrow polydispersity indexes.98, 153, 154

1.6 Gold nanoparticles for lasing application A dye laser, consisting of an organic dye in liquid solution155 or in solid polymer

matrices156 as gain medium, are widely used for biosensing,157 medical treatment,158

12

and optics159 due to wide bandwidth suitable for tunable lasers and pulsed lasers, easy

fabrication, and low cost of the dyes. A high energy source of light, e.g., an external

laser, is needed to "pump" the liquid beyond its lasing threshold. But the problem with

dye molecules is their photobleaching under laser irradiation, which results in the

unstable output intensity and shortened operation time. Noble metal NPs were found

to have strong effects on fluorescence intensity of organic dyes, i.e., fluorescence

quenching160 or enhancement,161 by means of their LSPR located in the same range of

absorbance and fluorescence bands of dyes and dye–metal distances. Besides

fluorescence enhancement, many efforts have been put on finding effective

approaches to improve the photostability organic dyes, another critical property of the

dye laser, e.g., introducing a spacer shell between metal core and dye molecules,162 or

incorporating dye molecules into transparent solid matrices.163


2 Experimental methodology Methods used for paper I–X are described in detail in the respective “Materials

and methods” sections. Below follows an overview of each method with references to

the papers in which they are used.

2.1 Synthesis of inorganic nanoparticles

2.1.1 SPIONs (paper II, III, V, VI)

Monodisperse SPIONs were synthesized using thermal decomposition method,138

which include synthesis of iron-oleate complex from sodium oleate and FeCl3·6H2O

as first step and followed by refluxing the complex in octyl ether at ca. 297 °C for 1.5

h in the presence of oleic acid.

2.1.2 Mesoporous silica (paper II, III, IV, IX)

MCM-41 NPs was synthesized by adding TEOS to mixture of H2O, CTAB, and

NaOH with molar composition of 2000 H2O: 0.125 CTAB: 0.3 NaOH: 1 TEOS at

80 °C and stirred for 2 h followed by calcination at 600 °C for 12 h.

2.1.3 Gd2O(CO3)2·H2O and Gd2O3 (paper IV)

Gadolinium oxide carbonate hydrate [Gd2O(CO3)2·H2O] NPs were prepared by a

modified method, where aqueous solution of Gd(NO3)3·6H2O and urea were heated to

86 ºC under N2 for 2.5 h.164 Obtained Gd2O(CO3)2·H2O was calcined at 800 ºC in air

for 3 h to form gadolinium oxide (Gd2O3) particles.

2.1.4 Au nanocrystals with different morphologies (paper I, IV,

VI, VII, VIII, IX)

Gold NPs with spherical morphology were prepared using different methods. A

modified Brust two-phase method126 was used to prepare hydrophobic gold NPs,

where aqueous hydrochloroauric acid (HAuCl4) and chloroform containing TOAB

was stirred vigorously followed by addition of 1-dodecanethiol and aqueous solution

of sodium borohydride (NaBH4). After stirring for 3 h, the layer of chloroform

containing Au NPs was collected. Hydrophilic OLA-stabilized gold NPs were

14

synthesized according to a reported method,165 by adding OLA (5-200 mL; 70 %,

technical grade) to aqueous HAuCl4 (1 mM) at 80 °C. Alternatively, gold NPs with

spheroidal shapes were prepared by reducing HAuCl4 using ascorbic acid and NaBH4

at room temperature, in the presence of CTAB and silver nitrate (AgNO3).

High AR (average value of AR = 10–26) gold NRs were prepared by reducing the

growth solution of HAuCl4 and CTAB using ascorbic acid and NaBH4 in the presence

of different concentrations (0–50 mM) of HNO3 under ambient temperature. The

procedure for synthesis of low AR (ca. 4.5) gold NRs 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 NRs.166 Ultrathin gold nanowires with diameter of ca. 2

nm and length of a few micrometers were obtained as by-product when HAuCl4 was

reduced using OLA (32 equivalents with respect to gold salt) at 80 °C for 2 h.

2.2 Surface modification and functionalization In order to render nanomaterials with certain properties, e.g., hydrophilicity,

bio-inertness, antibiofouling, or as an intermediate layer for hierarchical structures,

surface of NPs were modified with specific molecules or crystals via chemical

bonding or hydrophobic interaction.

2.2.1 Coating Fe3O4 and Au with amorphous (mesoporous)

silica (paper II, III, IV, V, IX)

Oleic acid-capped Fe3O4 NPs were transferred to the water phase by mixing with

aqueous CTAB solution at 65 ˚C and then evaporated chloroform. To coat mSiO2, the

aqueous Fe3O4 suspension was diluted with water and the pH was tuned to 12 by the

addition of NaOH. The solution was heated to 70 ˚C and a specific amount of TEOS,

and corresponding four times of EtOAc, were slowly added to the reaction solution in

sequence (detailed concentrations of reagents in paper III). Gold NRs capped with

CTAB were coated with mSiO2 (AuNR@mSiO2) by the similar procedure. While, the

CTAB-capped spheroidal Au NPs for laser photostability experiment were coated


with silica at room temperature, by adding TEOS to Au NPs suspension in mixture of

water and ethanol at pH = 12.

Aforementioned hydrophilic gold NRs and hydrophobic gold NPs or QDs are

coated with common amorphrous silica using a general method. The aqueous gold

NRs suspension, gold NPs or QDs dispersed in chloroform were conducted with

centrifugation and redispersed in ethanolic mercaptocarboxylic acid, such as

3-mercaptopropionic acid (MPA) or MUDA, mixing for 2 h. Next, Stöber method was

used to grow amorphous silica on the surface of these NPs, which was redispersed in

mixture of water and ethanol followed by addition of ammonium and TEOS at room

temperature. On the amorphous silica coating, another layer of mSiO2 can be formed

by introducing ammonium, aqueous CTAB, and TEOS in sequence.

2.2.2 Coating Gd2O(CO3)2·H2O on mesoporous silica (paper IV)

Different amount of gadolinium stock solution containing Gd(NO3)3·6H2O and

urea was added to aqueous suspension of AuNR@mSiO2 for varied coating thickness.

The mixture was heated to 80 °C under N2 and stirred for 2.5 h. Samples for phantom

images were prepared by solidating AuNR@mSiO2@Gd2O(CO3)2·H2O suspension in

10 mL of 3 % agarose gel. Free Gd3+ ions leached from composite NPs in aqueous

suspension was quantified by spectrophotometric method based on the differences in

the visible spectra of free and complexed xylenol orange dye.167 The free Gd3+

concentration is directly proportional to the ratio of the absorbances at 573 and 433

nm. Over a given concentration range of free Gd3+ a linear dependence is observed.

Linearization of the experimental data yields the calibration equation, which is then

used for the determination of free Gd3+ concentrations.

2.2.3 Antibiofouling copolymer coating on Gd2O(CO3)2·H2O

(paper IV)

A random copolymer, poly(TMSMA-r-PEGMA-r-AAm/AAc), has been

synthesized via free radical polymerization of TMSMA and PEGMA and AAm or

AAc (with molar ratio of 2:1:1) in anhydrous THF. The solution was heated to reflux

under N2 and the polymerization was initiated by adding recrystallized AIBN. The

16

obtained copolymer was washed and freeze-dried for further use. Ethanolic

suspension of AuNR@mSiO2@Gd2O(CO3)2·H2O NPs and copolymer was heated to

reflux under N2 with stirring overnight. The pH value of aqueous suspension of

AuNR@mSiO2@Gd2O(CO3)2·H2O@poly(TMSMA-r-PEGMA-r-AAm) was tuned to

ca. 8.5 and conjugated with COOH-terminated FITC at room temperature.

2.2.4 Grafting thermo-sensitive copolymer from surface of

mesoporous silica (paper II)

Thermo-sensitive copolymer poly(NIPAAm-co-AAm) was coated on Fe3O4@

mSiO2 NPs via grafting-from strategy. The Fe3O4@mSiO2 NPs were functionalized

with TMSMA first and then TMSMA-modified Fe3O4@mSiO2 NPs were used as the

seeds to conduct free radical copolymerization on the surface of NPs. Typically,

monomers of NIPAAm and AAm and cross-linker MBA are mixed with aqueous

suspension of TMSMA-modified Fe3O4@mSiO2 NPs at 70 ˚C under a N2 flow before

the initiator potassium persulfate (KPS) is introduced.

2.3 Gold nanoparticles in laser gain media (paper VII, VIII,

IX) The sample for photostability experiment was prepared by mixing Rh6G aqueous

solution with same amount (defined as Au = 100 %) or 1 % (Au = 1 % of Rh6G)

number of hydrophobic gold NPs in chloroform using ultrasonic sonication. The

experimental setup and information of pump laser are detailed in paper VII.

Hydrophilic CTAB-capped Gold NPs and silica coated Au NPs were also examined

for photostability. Different concentrations of gold or Au@SiO2 NPs were mixed with

aqueous solution of Rh6G (see details in paper VIII and IX). The laser performance

test was similar with that for hydrophobic Au NPs.

2.4 Characterizations The morphology of NPs or thin films was characterized by JEOL JEM-2100F

field emission TEM (FETEM) operating at an accelerating voltage of 200 kV, or by

Zeiss Ultra 55 SEM at 3 kV, or by AFM (Veeco/Digital Instruments). Dark filed


images were obtained by high angle annular dark field (HAADF) scanning

transmission electron microscopy (STEM). Energy dispersive X-ray spectroscopy

(EDX) in JEM-2100F FETEM was used to analyze the elemental compositions.

Optical absorbance/transmittance spectra of particle suspension or thin films were

recorded by PerkinElmer Lambda 750 UV/Vis/NIR or Varian Cary 100 Bio UV-Vis

Spectrophotometer. Fluorescence spectra and images were captured by PerkinElmer

fluorescence spectrometer and IVIS optical imaging system, respectively. Raman

spectra were obtained using HORIBA Raman system. The PANalytical XPert Pro

powder diffractometer with Cu-Kα radiation (45 kV, 35 mA) was used to record XRD

patterns. Nitrogen sorption isotherms were measured on a Micromeritics ASAP 2020

pore analyzer at 77 K under continuous adsorption conditions. The hydrodynamic

diameter and surface charge of the particles in suspension were measured by

DelsaNano zetasizer (Beckman Coulter) or by Malvern Zetasizer. Concentrations of

elements in particle suspensions were measured by Thermo Scientific iCAP 6500

series ICP-AES or by Varian AAS. The thermal behavior of NPs was evaluated by

DSC-Q2000 and TGA-Q500 (TA instruments). A tunable, pulsed Q-switched

Nd:YAG laser with repetition rate of 40 Hz and pulse duration of 2.2 ns and a 532 nm

pulsed Nd:YAG laser with repetition rate of 20 Hz and pulse duration of 3 ns were

used for laser irradiation experiments. The filed dependent magnetizations were

measured 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.

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. MRI phantom images were

taken by a Siemens Trio MR Scanner (Siemens, Erlangen, Germany) at 3T. CT

scanning was performed with a 64-multirow detector CT equipment (General Electric

Light Speed VCT XT, GE Healthcare, Milwaukee, Wisconsin, USA) using 120-kVp

protocol and automatic dose modulation. All images were reformatted to 5 mm thick

coronal slices with 2.5 mm reconstruction overlap.

18

2.5 Biocompatibility evaluation

2.5.1 Cells/cell lines and culture conditions (paper I, III, IV, V)

Human peripheral blood mononuclear cells (PBMC) from healthy blood donors

were separated from buffy coats. MDDCs were generated in the presence of IL-4 and

GM-CSF as previously described.168 At day 6 of culture, the immature MDDCs

(4×105 cells/mL) were co-cultured with gold NRs for 24 h. The morphology of

MDDCs incubated with gold NRs were examined by Tecnai 10 TEM at 80 kV. The

phenotype of MDDCs was evaluated with flow cytometry analysis. MDDCs were

labeled with fluorescent phycoerythrin (PE) conjugated mouse monoclonal antibodies

(mAbs) and the fluorescence was measured with a FACSCalibur flow cytometer. Ten

thousand cells were counted and analyzed by the program CellQuestPro.

OC-k3 cells were cultured at permissive conditions: 33 °C, 10 % CO2 in DMEM

supplemented with 10 % FBS, 50 U/mL of recombinant mouse interferon-γ (IFN-γ)

and without antibiotics.169

A549 human lung adenocarcinoma epithelial cells (obtained from the American

Type Culture Collection) were maintained in DMEM 41965 supplemented with1 mM

sodium pyruvate, (100 U/mL penicillin-100 g/mL streptomycin), 10% fetal bovine

serum, and grown in 5% CO2 and 95% relative humidity at 37 °C.

2.5.2 Cell viability assay (paper I, III, IV, V)

To investigate the effect of gold NRs 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.170

OC-k3 cell viability was measured by flow cytometry analysis and MTS assay.

For flow cytometry analysis, OC-k3 cells were grown in 6-well plates at a density of

150000 cells/mL (2 mL in each well). After cells adhered to the plates (16–18 h), they

were treated with Fe3O4-CTAB or Fe3O4@mSiO2 NPs of various concentrations and

incubated for 3–48 h. After 3 and 48 h, respectively, the cells were recovered using


trypsin/EDTA solution (1 minute at 37 °C), which were then processed for analysis

using FACSCalibur flow cytometer. The samples were collected in the appropriate

tubes and 2 μg/mL PI was added. For MTS assay, the CellTiter 96 assay was used and

OC-k3 cells were grown in 96-well plates at a density of 5000 cells/mL (0.5 mL in

each well). After cells adhered to the plates (16–18 h), they were treated with varying

particle concentrations and incubated for 3 h. MTS is bio-reduced by the

dehydrogenase enzymes found in metabolically-active cells into a formazan product.

The quantity of formazan product measured by the absorbance at 492 nm is directly

proportional to the number of living cells in culture. Optical density (OD) was read at

492 nm in a microplate reader Sirio. Data are indicated as percentage of untreated

controls. All experiments were repeated three times and the results were reproducible.

Short-term A549 cell viability was determined by the MTT assay, which reflects

the mitochondrial function of cells. Briefly, cells were seeded at a density of 1x104

cells/well in a 96-well plate, and after 24 h culturing, the medium was changed to

DMEM without phenol red and serum. Three replicates were used for each sample of

AuNR@mSiO2@Gd2O(CO3)2·H2O and three independent experiments performed.

After exposure, the supernatant was removed and cells were washed once with PBS

(pH 7.4). One hundred microliters (100 μL) of MTT solution (0.5 mg/mL) was added

and incubated for 3 h at 37 °C. Finally, 50 μL of DMSO was added to dissolve the

formazan crystals. MTT conversion was quantified by measuring the absorbance at

570 nm using the SpectraMax 250 spectrophotometer.

20

3 Results and discussions

3.1 Superparamagnetic MRI contrast agents (paper III)

3.1.1 Magnetic mesoporous nanoparticles

Figure 3.1a shows the TEM micrograph of oleic acid capped SPIONs dispersed in

chloroform with narrow size distribution and an average diameter of 12.0 nm (σ ≈

5.0 %). Inset HRTEM image in Figure 3.1a shows the single crystalline nature of

these hydrophobic particles. After phase transfer, aqueous SPIONs capped by CTAB

retained its morphology. The low magnification images of Fe3O4@mSiO2 NPs (25–95

nm in diameter) 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 an mSiO2 layer with about 2 nm wormhole-like mesopores. The optimized

concentrations of reagents for different thicknesses of mSiO2 coatings are summarized

in Table 2.1. The ratios between different reagents (e.g., [CTAB]/[Fe3O4] and

[H2O]/[CTAB]) are critical in producing well-defined core-shell structures.

Figure 3.1 TEM micrographs of (a) hydrophobic Fe3O4 core NPs, and (b) 50 nm, (c) 75 nm, and (d) 95 nm core–shell Fe3O4@mSiO2 NPs with the same core. The insets show the magnified images for each sample.


The high angle XRD pattern of Fe3O4@mSiO2 (Figure 2 in paper III) exhibit a

broad peak centered at 22˚(2θ) consistent with the amorphous nature of the mSiO2

coating and the characteristic peaks of magnetite structure are retained. Average

crystal size of Fe3O4 cores determined by Debye–Scherrer formula is 11.1 nm. N2

adsorption/desorption isotherms of Fe3O4@mSiO2 (Figure 3 in paper III) exhibit a

characteristic type IV isotherm as expected for mSiO2 material. The BET surface area

and the total pore volume of extracted 75 nm Fe3O4@mSiO2 NPs are 202 m2 g-1 and

0.29 cm3 g-1, respectively.

3.1.2 Magnetic and MR studies of Fe3O4@mSiO2 nanoparticles

None of the suspensions of Fe3O4-CTAB and Fe3O4@ mSiO2 NPs have shown a

hysteresis for magnetization curves (Figure 4 in paper III), demonstrating their

superparamagnetic property and suitability as MRI T2 contrast agents. The saturation

magnetization (Ms) of Fe3O4-CTAB, 50 nm and 75 nm Fe3O4@mSiO2 NPs are 48.7,

48.3, and 47.1 A m2 kg-1 Fe, and magnetic domain sizes are calculated as 11.7, 11.8,

and 11.8 nm by Langevin equation,105 respectively. The relaxivity values (r1 and r2)

for particles with various coating thicknesses are summarized in Table 3.1. The r1

values of Fe3O4@mSiO2 NPs are one order of magnitude lower than that for

Fe3O4-CTAB, which is proposed to be due to the different effects between CTAB and

mSiO2 coating layers on mobility of water molecules.171 For Fe3O4@mSiO2 NPs with

varying coating thickness, it is found that the values of r1 are found effectively

decreased by ca. 6 times (at 20 MHz) and 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 %. 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.172 The decreases of r2 are due to the weakened locally-generated

magnetic field by Fe3O4 cores.173 Consequently, the r2/r1 ratio increases as a function

of the coating thickness, which are ca. 21 and ca. 14 times higher for 95 nm

Fe3O4@mSiO2 than that of the two commercial iron oxide-based contrast agents (see

Table 3.1), indicating their high efficiency on T2 MR imaging.

22

Table 3.1 Mean hydrodynamic diameters and relaxivities 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 r1

r2

r2/r1

r1

r2

r2/r1 by TEM) diameter [nm]a

Fe3O4–CTAB (11 nm) 67 31.25 81.37 2.61 13.69 82.18 6.01 Fe3O4@mSiO2 (50 nm) 60 3.65 84.26 23.1 1.31 92.13 70.3 Fe3O4@mSiO2 (75 nm) 77 2.13 79.93 37.5 0.97 87.54 90.3 Fe3O4@mSiO2 (95 nm) 96 0.61 50.13 82.2 0.31 55.44 179 Feridex172 72 40 160 4 − − − Resovist174 65 25 164 6.2 − − − a mean value of volume distribution

3.1.3 Relaxometric properties

The relaxometric data can be related to the morphological and physical properties

of magnetic particles via previously proposed proton relaxivity theories.5 By fitting

the NMRD curves, the average magnetic core size and specific magnetization of

Fe3O4-CTAB NPs are obtained as 12.5 nm and 48.2 Am2 kg-1 Fe, respectively, close

to the results measured by magnetometry and indicating monodispersity of these NPs.

While, for 50 nm Fe3O4@mSiO2, the magnetite core size is calculated as 28 nm and

specific magnetization as 14.3 Am2 kg-1 Fe (Table 3 in paper III). We ascribe these

differences to the effect of mSiO2 on diffusive permeability of water molecules, since

the determination of magnetic crystal radius r and specific magnetization Ms by

NMRD are closely related to diffusion parameters (e.g., τD and D).175, 176 Therefore,

when the coating is permeable to water molecules, magnetometry and relaxometry

result in similar results of the diameter of the magnetic cores and magnetization. On

the contrary, when the coating is completely impermeable, the magnetic core size

measured by relaxometry should exhibit the same value with hydrodynamic size of

the core-shell particles. Magnetic core size of 50 nm Fe3O4@mSiO2 NPs obtained by

relaxometry is 28 nm, an intermediate value between that measured by magnetometry

and hydrodynamic size of core-shell NPs. This demonstrates that the mSiO2 coating is


partially permeable and the water molecules seeping across the coating are highly

constrained in its spread.

3.1.4 Cell viability assessment

After 3 h incubation, Fe3O4-CTAB NPs induced decreases in MTS levels at 75

and 150 ng/mL and showed high toxicity at higher doses (highly significant, p<0.001).

The treatments with varying sized Fe3O4@mSiO2 NPs (35–95 nm) did not induce

considerable toxic effects and the mortality rate was not significantly different from

the untreated cells (Figure 6 in paper III; data confirmed also by flow cytometry

analysis). After 48 h incubation, Fe3O4-CTAB NPs produced significant toxicity level

at all the doses tested, while the Fe3O4@mSiO2 NPs had no major negative impact on

cell viability (Figure 7 in paper III). Previous report showed that γ-Fe2O3 particles

became drastically toxic after 48 h of exposure through oxidative stress,177 however

we did not observed any significant toxicity from Fe3O4@mSiO2 NPs after this period

of time. It is well documented that high concentration of CTAB is not

biocompatible,178 thus we cannot exclude its toxic effect as coating on Fe3O4 particles.

3.2 Multimodal MRI, CT, and fluorescence agent (paper IV)

3.2.1 Morphological, structural, and elemental analyses

The TEM bright field and HAADF-STEM images of AuNR@mSiO2@Gd2O

(CO3)2·H2O NPs have shown in Figure 3.2, where the hierarchical structure is clearly

visualized. Different thicknesses of Gd2O(CO3)2·H2O have been prepared. The

Figure 3.2 (a) TEM bright field, (b) HAADF-STEM images of AuNR@mSiO2@Gd2O (CO3)2·H2O NPs, and (c) line scan showing the spectral emission from the constituent elements.

24

elemental composition of the core-shell NPs was determined using EDS technique

and a line scan showed the spectral counts corresponding to Au, Si, O, Gd (Figure

3.2c). The XRD pattern of AuNR@mSiO2@Gd2O(CO3)2·H2O NPs (Figure 2 in paper

IV) include several clear peaks which can be indexed to the structure of gold, and

some other non-pronounced peaks and a broad peak might be attributed to the thin

layer coating of Gd2O(CO3)2·H2O. Owing to its limited growth on the surface of

AuNR@mSiO2, the Gd2O(CO3)2·H2O coating shows a low level of crystallinity.

The Vis-NIR spectra showed red-shifts of longitudinal peaks for AuNR@mSiO2

@Gd2O(CO3)2·H2O nanostructures compared to Au NRs and AuNR@mSiO2. This

shift is due to an increase in the local refractive index of the medium surrounding the

gold NR after the formation of the mSiO2 and Gd2O(CO3)2·H2O shells. The

longitudinal absorbance peaks for AuNR@mSiO2@Gd2O(CO3)2·H2O NPs, i.e., 836

nm, 846 nm, and 853 nm for three thicknesses in this work, locate in NIR range.

3.2.2 MRI, CT and fluorescence imaging

The MRI and CT phantom images (Figure 3.3) of AuNR@mSiO2@Gd2O (CO3)2·H2O

show clearly the concentration-dependent contrast evolution. The magnetic

relaxivities r1 and r2 were calculated as 107 s-1 mM-1 and 65.4 s-1 mM-1 of Gd,

respectively, much higher than those for clinical contrast agent of Gadovist. The

signal of CT is from both gold and gadolinium and the intensity is proportional to the

concentrations of these two elements. The sample containing 1 mM Au and 42 mM

Gd has a CT intensity of 342 HU, higher than that of the clinically used Visipaque

with the same concentration of iodine (188 HU).

To prolong the in vivo circulation time and avoid non-specific cell uptake, poly-

Figure 3.3 MRI (upper) and CT (lower) phantom images of AuNR@mSiO2@Gd2O (CO3)2·H2O NPs.


Figure 3.4 Fluorescence images of poly(TMSMA-r-PEGMA-r-AAm)-FITC coated (top) and non-coated (bottom) AuNR@mSiO2@Gd2O(CO3)2·H2O NPs. (TMSMA-r-PEGMA-r-AAm) was grafted to the surface of AuNR@mSiO2@

Gd2O(CO3)2·H2O NPs. Carboxylic acid-terminated FITC was conjugated with the

primary amine in the co-polymer. Figure 3.4 demonstrates the high intensity of

fluorescence for polymer-FTIC coated hybrid NPs.

3.2.3 Biocompatibility evaluation

The cytotoxicity of hybrid NPs with different Gd2O(CO3)2·H2O shell thicknesses

has been evaluated using A549 cells and a cell viability assay that measures the

metabolic activity of mitochondria. Upon exposure to 1 to 200 μg/mL core-shell

particles, cell viability slightly decreased in a dose-dependent manner (Figure 4 in

paper IV). At the highest dose tested of 200 μg/mL, which is considerably higher than

any dose that would be applied in vivo, cell viability was only modestly impaired to

about 80 % of control. Moreover, cell viability was similar after 24 h and 48 h of

exposure to the hybrid core-shell particles. Additionally, the hybrid particles with thin

Gd2O(CO3)2·H2O coatings were found having a higher percentage of leached Gd3+

than the particles with thicker coatings after 4 days incubation, while the total

concentrations of Gd3+ ions are in a similar magnitude for all samples. This might be

the reason that AuNR@mSiO2@Gd2O(CO3)2·H2O NPs have similar biocompatibility

regardless of their different thicknesses of Gd2O(CO3)2·H2O coating and different

concentrations of released Gd3+ ions.

26

3.3 Multifunctional core-shell composite nanoparticles

(paper II)

3.3.1 Morphology, structure, and magnetic properties

Figure 3.5a shows TEM images of poly(NIPAAm-co-AAm) coated

Fe3O4@mSiO2 NPs with different thicknesses of polymer layers. Polymer-coated

composite particles were examined with N2 adsorption/desorption and exhibit a

characteristic type IV isotherm for mSiO2 structures (Figure 3 in paper II). Magnetic

measurement of these composite particles demonstrates their superparamagnetic

property with no hysteresis. The Ms value (Figure 2 in paper II) is 49.1 emu g-1 Fe or

1.13 emu g-1 of total weight of the composite (ca. 2.3 wt % of Fe), prepared from 3

mmole of NIPAAm and AAm monomers (90%:10% in molar ratio).

3.3.2 Thermo-responsive properties and manipulation of LCST

The thermo-responsive properties of polymer-coated composite particles were

studies by measuring the change of hydrodynamic particle sizes upon varied

temperatures using DLS. The hydrodynamic size of composite particles has a sharp

decrease from 29 °C to 37 °C due to the collapse of the PNIPAAm chain and is almost

no change till 45 °C. By fitting the variation of hydrodynamic size on temperature

change, phase transition temperature, i.e. LCST, is found at ca. 33 °C (Figure 4b in

paper II).

By detecting the endotherm and exotherm of phase transition using DSC, the

thermo-responsive properties and changes of LCST were further studied. It is found

that, when the cross-linking density is fixed, LCST of polymer-coated composite is

decreased for higher NIPAAm content than the lower one. This is consistent with the

previous reports that as the polymer chain contains more hydrophobic constituent,

LCST becomes lower.179 On the other hand, if total molar amount of monomers is

fixed, LCST can also be changed by adjusting the molar ratio between hydrophobic

and hydrophilic segments of the polymer. Figure 3.5b shows the variations of LCST

according to the molar percentages of AAm. We found that more AAm content


Figure 3.5 (a) TEM images of P(NIPAAm-co-AAm) coated Fe3O4@mSiO2 composite NPs. The polymer layer was visualized after positive stain with phosphotungstic acid; (b) LCST of composite NPs using different percentage of AAm in total 3 mmole of NIPAAm and AAm monomers; photographs of aqueous suspension of composite NPs under the influence of a magnetic field (300 mT) at (c) 20 ˚C and (d) 40 ˚C.

displays a higher LCST, which is contrary to the observation for PNIPAAm polymer

shell. These results also suggest that LCST can be accurately controlled, i.e., 2.0–2.5

˚C increase per 0.15 mmole AAm addition. Figure 3.5a shows that Fe3O4@mSiO2@

P(NIPAAm-co-AAm) NPs have a very weak interaction with external magnetic field

and are stable for a few hours, which is obviously not suitable for magnetic separation

applications. When temperature is raised above the LCST, the composite aggregates

and was collected by the external magnet, which can be an excellent alternative for

currently used magnetic microbeads for magnetic separation applications.

3.4 High aspect ratio gold nanorods (paper I)

3.4.1 Morphological and structural studies

The effects of concentrations of HNO3 (0.2-72 mM) on the growth of gold NRs have

been studied. The ARs of gold NRs firstly increase and then decrease with increased

Figure 3.6 TEM images of gold NRs prepared by using HNO3 with the following concentrations: (a) 0, (b) 1 mM, (c) 3 mM, and (d) 72 mM in the solution.

28

Figure 3.7 TEM images of gold NPs at growth duration of (a) 5 min, (b) 1 h in the presence of 1 mM HNO3; and at (a) 5 min, (b) 1 h in the presence of 72 mM HNO3. All scale bars in white are 5 nm. HNO3 concentration (Figure 3.6). The highest AR was achieved using 3 mM HNO3,

while spherical NPs were obtained at a concentration of 72 mM HNO3 (Figure 3.6d).

3.4.2 Particle growth mechanism studies

The growth mechanism of gold NRs with the impact from HNO3 has been

attemptedly elucidated by studying the crystal structure of gold NRs formed at

different growth stages using HRTEM. In presence of 1 mM HNO3, most of the

formed particles at 5 min were found to be twinned (AR ca. 1.2), indicated by the

crystal lattices pointing to different directions but with same lattice spacing (Figure

3.7a). The lattice spacing of these five-fold twinned NPs was found to be ca. 2.4 Å

close to that of Au (111) crystal plane. These gold NPs grew anisotropically into

rod-shaped particles with ARs of ca. 17 after 1 h (Figure 3.7b). For the NRs produced

with 72 mM HNO3, only single crystal (not twinned) spherical NPs were formed at

the initial stage (Figure 3.7c). Furthermore, these NPs grew isotropically, instead of

anisotropically, with their diameter increasing from ca. 7 nm to ca. 15 nm within 1 h

(insets of Figure 3.7c-d). Interestingly, about 6% (12 out of 200) of these particles

showed five-fold twinned decahedral shape (Figure 3.7b), but without growth into

rod-shaped particles.

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.180 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 micelles181 is

predominant over the oxidative dissolution of multiple twinned particles. 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.182 Therefore, the mechanism of growth of high AR gold NRs 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 NRs.

3.4.3 Biocompatibility evaluation

3.4.3.1 Viability studies

It is found that NRs with high AR seem not to have an impact on cell viability

(Figure 6a, b in paper I) similar to spherical gold NPs. In contrast, low AR gold NRs

at a concentration of 50 µg/mL induced a considerable decrease in viability, involving

mainly late apoptosis (programmed cell death) and necrosis (uncontrolled cell death).

Improved biocompatibility was achieved for the low AR rods using a larger

number of washing steps to reduce the amount of CTAB capped on gold NRs,

however it causes particle agglomeration. This suggests that the decreased viability

for low AR rods was not due to morphology, but rather due to higher concentrations

of CTAB. We propose that the addition of HNO3 for high AR rods changes the

equilibrium of the chemical reaction and facilitate the removal of CTAB molecules

from the surface of high AR gold NRs compared with the low AR ones.

3.4.3.2 Immune modulatory effects

The immune modulatory effects of the high AR gold NRs on MDDCs were

evaluated, and results showed that the rods did not have any impact on the overall

expression of CD40, CD80, CD83 or CD86 on MDDCs (Figure 7 in paper I).

Together with the previous study on spherical gold NPs,168 we can therefore conclude

that neither the high AR NRs nor the spherical NPs induced any pronounced

30

maturation of the MDDCs, thus different morphologies of gold NPs did not seem to

cause any different cellular responses.

3.4.3.3 Cellular internalization study

The interaction between high AR rods and MDDCs has also been investigated via

examining the internalization of gold NRs by MDDCs using TEM. Within 1 h of

co-incubation with NRs (50 µg/mL), it could be detected that these NRs were inside

the cells and packed within vesicular structures, probably endolysosomes.183 After 24

h most cells contained high amounts of NRs (Figure 8 in paper I) with a similar

uptake as for 1 h, demonstrating an efficient uptake by MDDCs. The relationship

between the surface coating of gold NRs and cellular uptake has been studied

previously, and it was found that the binding of serum proteins plays a significant role

in NRs uptake.184 However, the MDDC uptake of the gold NRs in this study needs

further investigation.

3.5 Effects of gold nanoparticles on lasing performance

(paper VII, VIII, IX)

3.5.1 Gold nanoparticles and Au@SiO2

Small-sized, hydrophobic TOAB-capped Au NPs are shown in Figure 3.8a with

particle size of ca. 5.5 nm. The HRTEM image reveals their single crystalline nature

with the lattice spacing of ca. 2.0 Å, close to that of the Au (100) lattice plane. Figure

3.8b shows that multiple gold particles were coated together with 20 nm thick aSiO2

layer, and another layer of 15 nm thick mSiO2 can be grown on aSiO2 (Figure 3.8c).

The characteristic SPR peak of Au NPs is centered at 520 nm (Figure 3.8d). The

Figure 3.8 TEM images of (a) hydrophobic TOAB-capped Au NPs, (b) AuNP@aSiO2, (c) AuNP@aSiO2@mSiO2, and (d) visible absorbance spectrum of TOAB-capped Au NPs.


Figure 3.9 TEM images of (a) CTAB-capped Au NPs, (b) AuNPs@SiO2, and (c) visible absorbance spectrum of Au NPs and AuNPs@SiO2. hydrodynamic size of Au/Rh6G mixture is larger than Au NPs suspension, which

indicates the formation of Au/Rh6G assemblies, and the size increase is due to the

attached dye molecules.

To examine the size effect of Au NPs for lasing application, a bigger-sized (>10

nm) Au NPs were synthesized. Figure 3.9a shows that CTAB-capped Au NPs are

spheroidal shape with ca. 25 nm diameters. They were then coated with ca. 15 nm

mSiO2 layer directly using CTAB as the template. The LSPR absorbance peak of

AuNP-CTAB is at ca. 531 nm, which overlaps well with the peak of Rh6G. While,

AuNP@mSiO2 has the peak with a slight red-shift at 534 nm (Figure 3.9c).

Gold NPs are also synthesized via a facile method using OLA as capping agent

and reducing agent simultaneously. The concentration of OLA was found affecting the

morphology and formation of particles. Figure 3.10a shows the image of ca. 60 nm Au

NPs produced in presence of low concentration OLA. When the concentration of OLA

increases, i.e. in Figure 3.10b, small particles with diameters of ca. 10 nm were

formed and the by-product are a few micrometers long and 2 nm wide ultrathin gold

nanowires. In this work, the OLA-capped Au NPs has been examined by TGA and the

Figure 3.10 TEM images of (a) 60 nm OLA-capped Au NPs, (b) mixture of 10 nm Au NPs and nanowires, and (c) TGA spectrum of OLA-capped Au NPs.

32

Figure 3.11 Dependence of output intensity on the number of pulses in (a) un-normalized scale and (b) normalized scale for the gold/dye assemblies with Au = 100 % and 1 % of the number of dye molecules, respectively. result (Figure 3.10c) shows a two-step weight loss, which indicates the bilayer

arrangement of OLA molecules on the surface of Au NPs.

3.5.2 Influence of particle concentration and size

For small-sized Au NPs (ca. 5.5 nm), the fluorescence quenching of gold/dye

solutions with different Au NPs concentrations was compared (Figure 3.11). A high

particle concentration results in a lower output intensity during the whole processes

(Figure 3.11a), indicating a lower radiative rate of the dye. A normalized plot reveals

that a higher NP concentration gives a higher photobleaching rate at the beginning of

the measurement (Figure 3.11b), showing that the presence of small-sized Au NPs

speeds up the photobleaching of the dye molecules.

For bigger-sized Au NPs (ca. 25 nm), the laser output intensity clearly depends on

the Au NPs concentration in the samples (Figure 3.12). Compared to the sample with

Figure 3.12 Laser output intensity as a function of the number of pump pulses for samples of dye mixed with different concentration of gold NPs (details in paper VIII). The output intensity of each sample is normalized to the maximum intensity from the pure dye (N0).


pure dye solution, samples with relatively low gold NPs concentrations (paper VIII)

exhibit enhancement for the lasing intensity, with maximum enhancement of 54 %. It

is likely that the observed enhancement is a combined effect of both pumping field

enhancement and fluorescence field enhancement. When the concentration increases

more (Figure 3.12b), the enhancement factor is smaller than 1 (quenching). The

photobleaching time monotonically decreases with the increase of gold NP

concentration, which indicates energy transfer from dye molecules to gold NPs.

3.5.3 Influence of surface coating

To suppress the energy transfer and prolong the bleaching time, charge-isolated

coatings, e.g. SiO2, can be applied to the gold NPs. Silica-coated Au NPs with

relatively low gold NPs concentration have also shown the enhancement of laser

output (Figure 3.13a), but the maximum enhancement factors are lower than those for

uncoated samples. It is also found that samples with Au@SiO2 show a distinct

prolongation of the photobleaching time over those with uncoated Au NPs. When

particle concentration increases, the output intensity of the cavity is quenched rather

than enhanced (Figure 3.13b). However, samples with Au@SiO2 shows approximately

the same intensity level as samples with uncoated Au NPs. And the photostability of

the gain medium is noticeably enhanced when gold NPs are coated with silica. The

photobleaching time for all samples with Au@SiO2 is almost the same, which

demonstrates concentration independent feature for the photostability of dye solution.

Figure 3.13 Laser intensity as a function of the number of pulses for the laser with samples of dye mixed with uncoated and coated gold NPs, (details in paper IX) and the control sample (N0) as the gain medium. The spectrum for each sample is normalized to peak value of N0.

34

4 Conclusions

This thesis presents my endeavour to develop functional inorganic and polymeric

nanomaterials through various methodologies and demonstrate their applications in

different fields, such as biomedicine and dye lasing. The challenge of this work is to

design the fabrication methods and assembly of different materials to obtain the

desired physical and chemical properties for specific applications.

A high-performance MRI T2 contrast agent has been developed based on uniform

Fe3O4@mSiO2 NPs with tunable coating thickness. The magnetic relaxivities (r1 and

r2) vary with the mSiO2 coating thickness. Enhanced r2/r1 ratios are found as a

function of the coating thickness, which is 20 fold higher than that of the commercial

contrast agents, indicating their excellent contrast enhancement property. This

evolution can be attributed to the effects of mSiO2 coating on water permeability to

the iron oxide core, according to the relaxometric studies.

A multifunctional nanomaterial has been prepared by grafting a thermo-

responsive copolymer as the outermost shell on Fe3O4@mSiO2 NPs. In addition to the

superparamagnetic property for T2 MRI, porous structure for loading, the phase

transition behaviour of this material for temperature controlled separation or valving

can be finely adjusted by tuning the composition of copolymers. The integrated

capabilities from different components endow this material high potential for future

medical diagnostic and therapeutic applications.

Hierarchical multicore-shell structured nanomaterials were fabricated for

multimodal MRI, CT, and fluorescence imaging. It is possible to grow mSiO2 directly

on CTAB-capped Au NRs and then deposit Gd2O(CO3)2·H2O on mSiO2. The intimate

contact with protons for gadolinium and strongly absorbing X-ray for both gold and

gadolinium result in enhancement of MRI and CT contrast compared with the clinical

contrast agents. Further PEGylation allows the increase of circulation time and can be

conjugated with FITC for fluorescence imaging.

High AR gold NRs have been prepared in presence of HNO3 using a non-seeded


method. The growth mechanism has been explained based on the effects of HNO3 on

elongation of micelles and oxidative dissolution of Au(0) together with Ostwald

ripening. High AR gold NRs are found no impact on the viability of primary MDDCs

and no significant effect on their cell surface molecules involved in T cell activation.

Both spherical gold NPs and high AR gold NRs did not seem to cause any different

cellular responses.

Gold NPs have been investigated on their capability for the modification of

fluorescence and photostability properties of gain media used for organic dye laser.

Small-sized gold NPs with diameter of ca. 5.5 nm are found to be detrimental to the

photostability of the gain media because the NPs offer extra channels of nonradiative

decay by energy transfer. Bigger-sized gold NPs with diameter of ca. 25 nm show the

similar degraded photostability of gain media with small sized NPs, even though

fluorescence enhancement can be achieved. To suppress the energy transfer from the

dye molecules to Au NPs for minimal photostability deterioration, a layer of SiO2 has

been coated on Au NPs. The results show that the photostability of gain media for

lasing applications has been enhanced.

36

Future work

Materials scientists always have ambitions to improve the functionality and

efficacy of materials used for various applications. For biomedical application,

nanoparticulate materials show superior performance than the currently used clinical

agents. To minimize the administration frequency, an all-in-one agent with

simultaneous diagnostic and therapeutic functions is desired. This agent should not

induce any major toxic or immune effects, and can deliver the payloads to

pathological sites and release them in a controlled way. Some potential efforts of

using nanomaterials for biomedical applications are suggested as follows.

Thermo-sensitive polymer coated Fe3O4@mSiO2 NPs presented in this thesis can

be move one step towards magnetically-triggered drug release. The drug can be

loaded at high temperature globule state and retained in cavity of mSiO2 at low

temperature coil state. A high-frequency magnetic field (HFMF) can be applied and

the rotation of magnetic NPs generate heat energy. Subsequently, drug release is

accelerated by controlled bursting to a therapeutically effective concentration.

For selective therapy, Fe3O4@mSiO2 NPs can be coated with pH-sensitive

polymers and targeting moiety. It can be used to load anticancer drug and will only

release the payload when reaches the pathological sites with low pH values.

To improve the synthetic method for multimodal imaging agent, ligand exchange

strategy can be applied to Au NRs by replacing CTAB with mercaptocarboxylic acid,

and then directly coat them with a layer of gadolinium oxide carbonate.

A new multifunctional platform for simultaneous imaging and therapy can be

realized by grafting upconversion fluorophore, e.g., lanthanide doped nanocrystals, on

gold NRs. With excitation in the NIR region, pconversion fluorescence imaging can

be used for imaging of biological cells and tissues. Meantime, cell destruction is also

possible, if the energy of NIR laser is high enough.

Gold NPs and NRs can be useful owing to their optical properties for

photodynamic therapy (PDT), which is a method for clinical treatment by destroying

diseased cells and tissues using a combination of light and photosensitizing drugs.


Acknowledgements Firstly, I am profoundly grateful to my supervisor, Prof. Mamoun Muhammed,

for leading me into the world of academic research and the interesting field of nanotechnology. I am always been encouraged to explore novel solutions for better performance. His enlightening ideas and kindness are always strong supports during my study and are bound to inspire me in my future career.

I would like to thank my co-supervisor Assoc. Prof. Muhammet Toprak for his great help from research to everyday life, especially for his kind help in revising my manuscripts. His continuous encouragement and guidance are highly appreciated. I wish him a great success in the scientific career and a happy family.

I want to thank Dr. Jian Qin for his great patience, genius ideas and professional guidance on my first step of research. I really learn a lot from him and believe he will continue to succeed for professional career. Sincere thanks to Dr. Shanghua Li and Dr. Andrea Fornara for the help, encouragement, and invaluable advices during these years. I wish them a splendid future, as well for the family. I am very grateful to my colleagues and friends in Functional Materials Division, Dr. Abdusalam Uheida, Dr. Xiaodi Wang, Dr. Ying Ma, Dr. Abhilash Sugunan, Sverker Wahlberg, Terrance Burks, Mohsin Saleemi, Ramy El Sayed, Mazher Yar, Robina Shahid, Yichen Zhao, Mohsen Tafti, Nader Nikkam, Dr. Mohamed Eita, Dr. Marta Perez, Milad Yazdi, and all former colleagues, for their great friendship, help and support for my research. I appreciate Hans Bergqvist and Dr. Wubeshet Sahle for their great work of maintaining and helping on use of electron microscopes.

I would like to thank Lin Dong and Assoc. Prof. Sergei Popov from Optics Division, KTH, for the great collaboration and papers. Many thanks to Dr. Jiantong Li and Prof. Mikael Östling from ICT, KTH, for the interesting and great work on graphene. I am grateful to my coworkers from Karolinska Institute for the fruitful collaborations: Dr. Helen Vallhov, Assoc. Prof. Susanne Gabrielsson and Prof. Annika Scheynius from Department of Medicine; Dr. Carmen Vogt and Prof. Bengt Fadeel from Institute of Environmental Medicine; Dr. Jingwen Shi and Prof. Lennart Möller from Department of Biosciences and Nutrition. Thanks to Dr. Torkel Brismar in Department of Radiology, Karolinska University Hospital, for generous help on MRI and CT imaging, and professional guidance. 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.

Last but not least, I would like to thank my parents for their endless support and encouragement. I wish to express my deep love to my wife Min Tian, for her unconditional love and support for my scientific career during these years. I also want to thank my parents-in-law for their kindness and support.

38

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Fei Ye PhD529974/FULLTEXT01.pdf · 2012. 5. 31. · iv 6G/gold nanoparticles: impact of SiO2...

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