Fei Ye Lic - kth.diva-portal.org

Synthesis of Nanostructured and Hierarchical Materials for

Bio-Applications

Fei Ye

叶飞

Licentiate Thesis

Stockholm 2011

Division of Functional Materials

School of Information and Communication Technology

Royal Institute of Technology

Postal address Division of Functional Materials

School of Information and

Communication Technology

Royal Institute of Technology

Electrum 229

Isafjordsgatan 22

SE 164 40, Kista, Sweden

Supervisor Prof. Mamoun Muhammed

Email: [email protected]

TRITA-ICT/MAP AVH Report 2011:04

ISSN 1653-7610

ISRN KTH/ICT-MAP/AVH-2011:04-SE

ISBN 978-91-7415-903-5

© Fei Ye, 2011

Kista Snabbtryck AB, Kista 2011

Functional Materials Division, KTH, 2011 i

Abstract In recent years, nanostructured materials incorporated with inorganic particles

and polymers have attracted attention for simultaneous multifunctional biomedical

applications. This thesis summarized three works, which are preparation of

mesoporous silica coated superparamagnetic iron oxide (Fe3O4@mSiO2)

nanoparticles (NPs) as magnetic resonance imaging T2 contrast agents, polymer

grafted Fe3O4@mSiO2 NPs response to temperature change, synthesis and

biocompatibility evaluation of high aspect ratio (AR) gold nanorods.

Monodisperse Fe3O4@mSiO2 NPs have been prepared through a sol-gel process.

The coating thickness and particle sizes can be precisely controlled by varying the

synthesis parameters. Impact of surface coatings on magnetometric and relaxometric

properties of Fe3O4 NPs is studied. The efficiency of these contrast agents, evaluated

by MR relaxivities ratio (r2/r1), is much higher than that of the commercial ones. This

coating-thickness dependent relaxation behavior is explained due to the effects of

mSiO2 coatings on water exclusion.

Multifunctional core-shell composite NPs have been developed by growing

thermo-sensitive poly(N-isopropylacrylamide-co-acrylamide) (P(NIPAAm-co-AAm))

on Fe3O4@mSiO2 NPs through free radical polymerization. Their phase transition

behavior is studied, and their lower critical solution temperature (LCST) can be subtly

tuned from ca. 34 to ca. 42 ˚C, suitable for further in vivo applications.

A seedless surfactant-mediated protocol has been applied for synthesis of high

AR gold nanorods with the additive of HNO3. A growth mechanism based on the

effect of nitrate ions on surfactant micelle elongation and Ostwald ripening process is

proposed. The biocompatibility of high AR nanorods was evaluated on primary

human monocyte derived dendritic cells (MDDCs). Their minor effects on viability

and immune regulatory markers support further development for medical applications.

Keywords: Fe3O4, mSiO2, core-shell, MRI, multifunctional, PNIPAAm, LCST, gold,

nanorod, nitric acid, AR, MDDC, biocompatibility, immunomodulation

ii

Functional Materials Division, KTH, 2011 iii

LIST OF PAPERS

This thesis is based on following publications: 1. Fei Ye, Helen Vallhov, Jian Qin, Evangelia Daskalaki, Abhilash Sugunan,

Muhammet S. Toprak, Andrea Fornara, Susanne Gabrielsson, Annika Scheynius and Mamoun Muhammed, “Synthesis of high aspect ratio gold nanorods and their effects on human antigen presenting dendritic cells”, in press, International Journal of Nanotechnology, 2011

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

“Multifunctional core-shell nanoparticles: superparamagnetic, mesoporous, and thermo-sensitive”, in press, Journal of Nanoparticle Research, 2011, DOI: 10.1007/s11051-011-0272-8

3. Andrea Kunzmann, Britta Andersson, Carmen Vogt, Neus Feliu, Fei Ye, Susanne

Gabrielsson, Muhammet S. Toprak, Tina Thurnherr, Sophie Laurent, Marie Vahter, Harald Krug, Mamoun Muhammed, Annika Scheynius and Bengt Fadeel, “Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells”, in press, Toxicology and Applied Pharmacology, 2011, DOI: 10.1016/j.taap.2011.03.011

Other work not included: 1. Andrea Fornara, Alberto Recalenda, Jian Qin, Abhilash Sugunan, Fei Ye, Sophie

Laurent, Robert N. Muller, Jing Zou, Abo-Ramadan Usama, Muhammet S. Toprak and Mamoun Muhammed, “Polymeric/inorganic multifunctional nanoparticles for simultaneous drug delivery and visualization”, Materials Research Society Symposium Proceedings Vol. 1257, 2010, 1257-O04-03

2. Lin Dong, Fei Ye, Jun Hu, Sergei Popov, Ari T. Friberg and Mamoun Muhammed,

“Fluorescence quenching and photobleaching in Au/Rh6G nanoassemblies: impact of competition between radiative and non-radiative decay”, Journal of the European Optical Society − Rapid Publications, 2011, 6, 11019

3. Fei Ye, Sophie Laurent, Jian Qin, Alain Roch, Muhammet S. Toprak, Robert N.

Muller and Mamoun Muhammed, “Uniform mesoporous silica coated iron oxide nanoparticles as highly efficient MRI T2 contrast agents with tunable proton relaxivities”, Manuscript

iv

Conference presentations 1. Fei Ye, Jian Qin, Muhammet S. Toprak and Mamoun Muhammed,

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

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

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

3. Fei Ye, Jian Qin, Sophie Laurent, Muhammet S. Toprak, Robert N. Muller and

Mamoun Muhammed, “Uniform mesoporous silica coated superparamagnetic iron oxide nanoparticles for T2-weighted magnetic resonance imaging”, 12th Bi-Annual Conference on Contrast Agents and Multimodal Molecular Imaging, May 19-21, 2010, Mons, BELGIUM (poster)

4. Andrea Fornara, Alberto Recalenda, Jian Qin, Abhilash Sugunan, Fei Ye, Sophie

Laurent, Robert N. Muller, Jing Zou, Abo-Ramadan Usama, Muhammet S. Toprak and Mamoun Muhammed, “Polymeric/inorganic multifunctional nanoparticles for simultaneous drug delivery and visualization”, Materials Research Society Spring meeting (2010 MRS), Apr 5-9, 2010, San Francisco/California, USA

5. Carmen Vogt, Andrea Kunzmann, Britta Andersson, Fei Ye, Neus Feliu Torres,

Tina Thurnherr, Sophie Laurent, Jean-Luc Bridot, Robert Muller, Muhammet Toprak, Harald F. Krug, Annika Scheynius, Bengt Fadeel and Mamoun Muhammed, “Tunable superparamagnetic Fe3O4-SiO2 core-shell nanoparticles: synthesis, characterization, and in vitro compatibility with immune-competent cells”, 34th International Conference and Exposition on Advanced Ceramics and Composites, Jan 24-29, 2010, Daytona Beach, Florida, USA

6. Lin Dong, Jun Hu, Fei Ye, Sergei Popov, Ari T. Friberg, Mamoun Muhammed,

“Influence of nanoparticles concentration on fluorescence quenching in gold/Rhodamine 6G nanoassemblies”, 2009 Asia Communications and Photonics Conference and Exhibition, Nov 2-6, 2009, Shanghai. CHINA

7. Fei Ye, Abhilash Sugunan, Jian Qin and Mamoun Muhammed, “A general method

for preparation of mesoporous silica coating on gold nanorods, nanoparticles, and quantum dots”, EuroNanoMedicine 2009, Sept 28-30, 2009, Bled, SLOVENIA (poster)

8. Stefan Gustafsson, Andrea Fornara, Fei Ye, Karolina Petersson, Christer

Johansson, Mamoun Muhammed and Eva Olsson, “TEM investigation of magnetic nanoparticles for biomedical applications”, in Materials Science, edited by Silvia Richter and Alexander Schwedt (14th European Microscopy Congress,

Functional Materials Division, KTH, 2011 v

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

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

gold nanorods”, Nanoimmune Workshop, Feb 13, 2007, Uppsala, SWEDEN

vi

Contributions of the author

Paper 1. Planning of experiments, preparing the materials, performing material characterization, evaluation of the results, and writing main part of the article. Paper 2. Planning of experiment, preparing the materials, performing material characterization, evaluation of the results, and writing the article. Paper 3. Preparing the materials, performing material characterization, and writing parts of the article.

Functional Materials Division, KTH, 2011 vii

ABBREVIATIONS AND SYMBOLS

AAm acrylamide AAS atomic absorption spectroscopy AR aspect ratio ATRP atom transfer radical polymerization BET Brunauer-Emmett-Teller CT computed X-ray tomography CTAB cetyltrimethylammonium bromide DSC differential scanning calorimetry EtOAc ethyl acetate FDA Food and Drug Administration HRTEM high resolution transmission electron microscopy ICP-AES

inductively coupled plasma-atomic emission spectrometry

IR infrared LCST lower critical solution temperature [˚C] LPS lipopolysaccharide M magnetization [emu g-1] MBA N,N’-methylene bisacrylamide MCM Mobil crystalline materials MDDC monocyte-derived dendritic cells MR magnetic resonance MRI magnetic resonance imaging NIPAAm N-isopropylacrylamide NIR near infrared NLDFT nonlocal density functional theory NMR nuclear magnetic resonance NMRD nuclear magnetic resonance dispersion NP nanoparticle PCS photon correlation spectroscopy PDLA poly(D,D-lactide) PEG poly(ethylene glycol) PET positron emission tomography PLGA poly(lactic acid-co-glycolic acid) PLLA poly(L,L-lactide) PNIPAAm poly(N-isopropylacrylamide) P(NIPAAm-co-AAm) poly(N-isopropylacrylamide-co-acrylamide) QD quantum dot RAFT reversible addition fragmentation chain transfer RES reticuloendothelial system RF radio frequency

viii

SAED selected area electron diffraction SPECT single-photon-emission computed tomography SPIO superparamagnetic iron oxide SPION superparamagnetic iron oxide nanoparticle SPR surface plasmon resonance TEM transmission electron microscopy TEOS tetraethyl orthosilicate TMSMA 3-(trimethoxylsilyl) propyl methacrylate TSC trisodium citrate VSM vibrating sample magnetometer XRD X-ray diffraction σ standard deviation

τD translational correlation time [s]

τN Néel relaxation time [s]

Functional Materials Division, KTH, 2011 ix

Table of contents

ABSTRACT…………………………………………………………………………...I

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

ABBREVIATIONS AND SYMBOLS…………………………………………….VII TABLE OF CONTENTS…………………………………………………………...IX 1 INTRODUCTION.................................................................................................1

1.1 OBJECTIVES ..............................................................................................................1 1.2 OUTLINE ....................................................................................................................2 1.3 MULTIFUNCTIONAL NANOSTRUCTURES ..........................................................3

1.3.1 Core-shell or micellar structure ............................................................................4 1.3.2 Porous structure ....................................................................................................5 1.3.3 Heterodimer or core-satellite structure.................................................................6

1.4 FUNCTIONAL NANOMATERIALS FOR BIOMEDICAL APPLICATIONS ..........7 1.4.1 Drug carriers ........................................................................................................7 1.4.2 Imaging probes......................................................................................................8 1.4.3 Thermo-therapy agents .........................................................................................9 1.4.4 Stimuli-sensitive drug delivery agents.................................................................10

1.5 BIOMEDICAL APPLICATIONS OF NANOMATERIALS .....................................11 1.5.1 Magnetic resonance imaging ..............................................................................11

1.5.1.1 Physical background ................................................................................11 1.5.1.2 Contrast agents .........................................................................................12 1.5.1.3 Superparamagnetism ................................................................................13 1.5.1.4 Preparation methods for magnetic NPs ....................................................13

1.5.2 Plasmonic photothermal therapy ........................................................................15 1.5.2.1 Surface plasmon resonance ......................................................................15 1.5.2.2 Preparation methods for gold NPs and NRs.............................................15

1.5.3 Temperature-triggered drug release....................................................................16 1.5.3.1 Thermo-responsive phase transition.........................................................16 1.5.3.2 Preparation methods for thermo-sensitive polymers ................................17

2 EXPERIMENTAL ..............................................................................................18 2.1 MAGNETIC MESOPOROUS NANOPARTICLES..................................................18

2.1.1 Synthesis of SPION and phase transfer...............................................................18 2.1.2 Fabrication of Fe3O4@mSiO2 .............................................................................18

2.2 PREPARATION OF THERMO-SENSITIVE COMPOSITE NANOPARTICLES...19 2.3 SYNTHESIS OF GOLD NANORODS.....................................................................19 2.4 CHARACTERIZATIONS..........................................................................................20 2.5 BIOCOMPATIBILITY EVALUATION.....................................................................21

2.5.1 Co-culture immature MDDCs with gold nanorods and spheres .........................21 2.5.2 Flow cytometry analysis......................................................................................21 2.5.3 Cell viability assay ..............................................................................................21

3 RESULTS AND DISSCUSSIONS .....................................................................22 3.1 SUPERPARAMAGNETIC MESOPOROUS CORE-SHELL NANOPARTICLES ..22

3.1.1 Morphology study ...............................................................................................22 3.1.2 Characterization of mesoporous silica................................................................24 3.1.3 Magnetometric and relaxometric properties .......................................................25 3.1.4 MR studies of Fe3O4@mSiO2 nanoparticles .......................................................26

3.2 MULTIFUNCTIONAL CORE-SHELL COMPOSITE NANOPARTICLES ............28 3.2.1 Morphological and structural studies .................................................................28

x

3.2.2 Magnetic properties ............................................................................................30 3.2.3 Thermo-responsive properties and manipulation of LCST .................................31

3.3 HIGH ASPECT RATIO GOLD NANORODS ..........................................................34 3.3.1 Morphological and structural studies .................................................................34 3.3.2 Particle growth mechanism studies.....................................................................35 3.3.3 Biocompatibility evaluation of high aspect ratio gold nanorods ........................38

3.3.3.1 Viability studies........................................................................................38 3.3.3.2 Immune modulatory effects......................................................................39 3.3.3.3 Cellular internalization study ...................................................................41

4 CONCLUSIONS .................................................................................................42

FUTURE WORK.......................................................................................................43

ACKNOWLEDGEMENTS ......................................................................................44

REFERENCES...........................................................................................................45

Functional Materials Division, KTH, 2011 1

1 Introduction

Nanotechnology, a concept first introduced by Richard Feynman during his talk

in December 1959,1 has implications from the medical, ethical, and environmental

applications, to many fields such as materials science, engineering, biology, chemistry,

physics, computing, and communications. Nanomaterials, feature in nanoscale size,

are key components for the development of nanotechnology due to the extraordinary

physical and chemical properties stemming from their nanoscale dimensions. Owing

to the similar size to that of most biological molecules and structures, nanomaterials

have been exploited by the biological and medical research communities for their

unique properties for various applications. Along with the tremendous development of

this research field, terms such as biomedical nanotechnology, nanobiotechnology, and

nanomedicine are coined to describe hybrid of the fields.2 Thus far, the integration of

nanomaterials with biology has led to numerous developments, such as diagnostic

devices, contrast agents, analytical tools, physical therapy applications, and drug

delivery vehicles, etc.3

1.1 Objectives

In this thesis, we are aiming to develop multifunctional NPs used in biomedical

applications, primarily as a contrast agent for MRI, temperature-responsive

biomaterials for magnetic separation and drug release, as well as evaluating the

biocompatibility of the nanomaterials.

1). SPION was synthesized in non-polar solvent and in order to use it as MRI

contrast agents, CTAB is employed as a phase transfer agent to render the dispersity

of SPIONs in aqueous phase. Mesoporous silica (mSiO2) coating layers have been

grown on surface of SPIONs via the organic template of CTAB. The dependence of

magnetic relaxivities and contrast enhancing properties of the core-shell NPs on the

thickness of the SiO2 coating layer are evaluated.

2). To incorporate thermosensitive property with core-shell magnetic

2

mesoporous NPs, for temperature-triggered magnetic separation or potential

temperature-controlled drug release applications, PNIPAAm is used for response to

temperature variation and a co-monomer of AAm for tuning this property.

3). As another type of nanomaterials for biomedical applications, gold nanorods

with high AR are prepared through a non-seeded and surfactant-mediated method.

Prior to further medical applications, the biocompatibility of the high AR gold

nanorods is evaluated by the viability and immune modulatory analyses on human

primary MDDCs.

1.2 Outline

This thesis deals with the development of multifunctional NPs for various

biomedical applications, such as MRI and magnetic separation, and potential

temperature-sensitive drug release, and thermal therapy.

Chapter 1.3 briefly introduces several frameworks for the construction of

hierarchical nanomaterials with simultaneous multifunctions, e.g. visualization,

environment-sensitivity, loading, targeting, and photothermal heating. Chapter 1.4

gives an overview on different categories of nanomaterials as drug carriers, e.g.

mesoporous materials, polymer particles, or liposomes; imaging and thermo-therapy

agents, e.g. iron oxide, or gold; and temperature-sensitive moieties, e.g. PNIPAAm.

Chapter 1.5 briefly presents the physical background of some biomedical

applications and the use of the functional nanomaterials.

Chapter 2.1 starts with the preparation methodology of SPIONs and mSiO2

coated Fe3O4 (Fe3O4@mSiO2) core-shell NPs. In Chapter 2.2, synthesis and grafting

methods of thermo-sensitive polymers are summarized. Synthesis of gold nanorods is

described in Chapter 2.3. Following characterization of physico-chemical properties

of materials by different techniques in Chapter 2.4, biocompatibility evaluation is

reported in Chapter 2.5.

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

In Chapter 3.1, the morphology and structure of Fe3O4@mSiO2 core-shell NPs with


variable coating thicknesses are discussed. The efficiency of these NPs as MRI T2

contrast agents are evaluated on the magnetic relaxivities and the impacts of mSiO2

coating thickness are also studied. In Chapter 3.2, thermo-sensitive polymer

PNIPAAm coated Fe3O4@mSiO2 NPs (designated as Fe3O4@mSiO2@PNIPAAm) are

studied with respect to magnetism, porosity, and thermal behaviors. LCST of

Fe3O4@mSiO2@PNIPAAm NPs can be manipulated by adjusting the composition of

the monomers for polymerization. Chapter 3.3 discusses the effect of nitric acid on

the crystal structures of the initially formed nuclei and consequently the growth of

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

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

which were compared with the effects caused by low AR gold nanorods.

1.3 Multifunctional nanostructures

Multifunctional nanomaterials, incorporating several components with different

functions into one entity, have attracted plenty of interest due to their combined

functionalities capable for simultaneously achieving multiple tasks, which can be

advantageously used as compared to components with a single function. Numerous

studies have been conducted on the development of multifunctional NPs as tools for

imaging,4-10 drug delivery,9-12 biosensing,13, 14 cancer therapy,5-8 and magnetic cell

separation.15 Obviously, a smart combination of the principles would lead to novel

tools with many applications in life sciences.

In some applications, appropriate designs of the nanostructures are required to

achieve specific functionalities. For example, multifunctional nanomaterials capable

to respond to external stimuli, such as changes of temperature,16 pH value,17

photoirradiation,18 ultrasound,19 biomolecules,20 and magnetic field,21 need sensitive

moieties exposed to or permeable to the external sources of excitation. In MRI

applications, in order to enhance T1 signals, paramagnetic electron spins of the T1

contrast agent are required to be directly in contact with water molecules via dipolar

interactions between electron spins of the contrast agent and nuclear spins of water.22,

4

23 PEG modification of NPs without affecting the characteristics of the NPs is also

necessary to avoid recognition by the RES prior to reaching targeting tissues.24

Biocompatibility is another key factor for the implants in medical applications, which

means good blood compatibility and low toxicity.25

1.3.1 Core-shell or micellar structure

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

bringing together different functional materials into one entity (Figure 1.1a). The core

normally is a solid NP of various materials such as metal,26-28 oxides,9, 29-31 or

polymers.32-34 In most of the cases, the functionalities of the core NPs are retained in

the presence of coating layers. In some other applications, the core particles act as a

sacrificial template for grafting or growing the shell materials with certain

morphology or functionality, and then are removed by dissolution,35 etching,36-38 or

decomposition through calcination39/UV irradiation40 in order to form cavities to load

drugs in the interior space. Alternatively, the sandwiched layer as the barrier of cores

also can be removed to facilitate the retention of physical properties of core

materials41 or the transmission of external stimuli to the core.42 Instead of solid cores,

the microemulsion method has been used to prepare nanostructures with soft cores of

oil beads or hydrophobic domain of polymers,43, 44 which contain hydrophobic interior

NPs or can be encapsulated with hydrophobic drugs. Liposomes45, 46 (Figure 1.1b) and

Figure 1.1 Schematic representations of multifunctional NPs with (a) a core-shell structure47 combined with targeting, imaging, cell-penetrating, stimulus-selective agents, therapeutic compounds, and a stabilizing polymer to ensure biocompatibility; (b) a liposome,45 and (c) a typical spherical micelle48 structure.


micelles48-51 (Figure 1.1c) with core-shell structures are also functional materials used

for therapeutic applications. The unique structure of liposome with hydrophilic

interior and hydrophobic lipid double layer facilitates the incorporation of hydrophilic

and hydrophobic drugs, respectively. They are protected from degradation and safely

transported until the liposome reaches the infected area, where the temperature is

higher than the body temperature and the structure is destabilized to release the

payloads.52 Micelles or reverse micelles are composed of monolayers of surfactant

molecules53 or amphiphilic polymers.48 Besides the manipulation of their

morphologies,54, 55 micelles with multifunctional modalities have been developed for a

variety of nanomedicine applications.56

1.3.2 Porous structure

Porous structure allows the integration of different materials in the pores, and it is

another type of system for simultaneous realization of multiple tasks. To arrange

multi-components in porous structures, NPs can be coated by porous materials (Figure

1.2a), or distributed inside the pores (Figure 1.2b). In the first construction, porous

layers are grown in situ around the NP cores along their surface-capping agents,

which may act simultaneously as organic templates for porous structures.9, 57 Or the

porous structure is formed by subsequent etching of the condensed shell materials.58

In another way, porous materials are template-synthesized utilizing surfactants,59

block copolymers,60, 61 or salts62 as sacrificial pore templates, and other materials can

be grown or loaded in the pores. Other methods, including replication of porous

alumina,63 liquid-crystal templates,64 or dealloying,65 to fabricate nanoporous metals

Figure 1.2 Schematic diagrams of porous multifunctional structures with (a) NPs as coated cores,9 or (b) NPs distributed in cavities of porous materials.66, 67

6

are reported. Very recently, non-toxic porous metal–organic frameworks have been

developed for biomedical applications.68

1.3.3 Heterodimer or core-satellite structure

Heterodimers are dumbbell-like NPs with two different functional NPs in

intimate contact (Figure 1.3a).69 They are commonly synthesized by sequential

growth of a second component on premade seeds through anisotropical nucleation

centered on one specific crystal plane of the seeding particles and their well-matched

lattice spacing lower the energy for epitaxial nucleation of second component. The

critical condition for growing heterodimmer NPs relies on promoting heterogeneous

nucleation of a second material while suppressing its homogeneous nucleation,70, 71

which results in the formation of separated NPs or core-shell structures. Other

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

crystal planes in dimer NPs, include immobilizing or coordinating a second

component with the substrate particles through chemical linkage to form core-satellite

morphology (Figure 1.3b).72-74 The incorporation of different functional materials

with controlled surface modifications into one unit is a promising approach for

designing multifunctional diagnostic and therapeutic applications.

Figure 1.3 Schematic illustrations of multifunctional (a) heterodimer NPs69 and (b)

NPs with core-satellite structures.72


1.4 Functional nanomaterials for biomedical applications

1.4.1 Drug carriers

Actively targeted drug carriers with entrapped drugs are capable of increasing the

therapeutic efficacy of a drug and reducing its systemic side-effects by delivering the

drug to the diseased site.75 Surface modification, e.g. folate modification, of the drug

carriers is necessary for targeting drug delivery.76, 77 Moreover, an ideal drug carrier

should not induce any immune response and should be degradable and produce

nontoxic degradation products. Hydrodynamic size of drug carriers is related with

their capabilities on overcoming the biological defense system and vascular barriers

for therapeutic delivery.78, 79 Furthermore, PEGylation of the drug carriers inhibits the

uptake by the RES and the blood circulation time increases.80

Drug carriers can be divided into two broad classes, depending on the interactions

between the drug and carrier, as macromolecular conjugates and particulate drug

carriers.81 In macromolecular conjugations, the drugs including proteins, antibodies,

and oligonucleotides are chemically linked to synthetic, natural, or pseudosynthetic

polymers. The chemical linker between the drug and the carrier is normally

acid-cleavable or reduction-sensitive, which will influence the controlled release of

the drug.82 Another category of drug carriers is particulate carriers that entrap the drug

in a loading space, thus isolating the drug from the environment and providing a high

degree of protection from enzymatic inactivation. Since covalent conjugation is not

necessary for entrapping the drug,83 a single carrier can be loaded with various drugs.

The most intensively investigated particulate drug carriers are liposomes and

polymeric NPs. Some of the medical applications of liposomes have reached the

preclinical stage and a few of liposomal formulations have been approved by FDA.84,

85 Among polymeric particles, biocompatible and biodegradable polyesters, such as

PLLA, PDLA, and PLGA, are widely studied.86 Hydrogels, another potential drug

carrier, can protect the drug from hostile environments and also control drug release

by changing the gel structures in response to environmental stimuli.87 Mesoporous

8

materials are also used as drug carriers with the features of high surface area/pore

volume and ordered pore network. Specifically, for mesoporous silica particles, their

silanol-containing surface can be functionalized for control of drug loading and

release.88-90

1.4.2 Imaging probes

Molecular imaging, with analytic and diagnostic ability at the molecular or

cellular level, has emerged recently which combines the molecular biology and in

vivo imaging.91, 92 Representative non-invasive imaging techniques include optical

imaging, CT, MRI, PET, SPECT, and ultrasound,93 which allow real-time

visualization of cellular functions of living organisms. Efforts have been devoted to

develop the imaging probe or contrast agent that can improve the sensitivity and

detectability of the current imaging tools for diagnosing disease,94 monitoring disease

progression,95 and tracking therapeutic response.96 Besides the available imaging

probes and contrast agents of organic dye,9 chelate,97 or radioactive agents,96 new

types of probes based on inorganic NPs have been developed, because of their useful

optical and magnetic properties derived from their compositions and nanometer

sizes.98 For instance, QDs show advantages in biological imaging, such as

size-tunable absorption and emission, flexibility in excitation due to broad and intense

absorption, and high fluorescence quantum yields even in the NIR wavelengths as

compared to organic dyes.99 SPION100, 101 are clinically available, relatively benign

contrast agents for MRI with good sensitivity and low toxicity. Metal-doped ferrite

NPs, e.g. superparamagnetic manganese ferrite (MnFe2O4),102 can induce significant

MR contrast-enhancement effects, especially useful for MRI of small cancers.103 Iron

oxides can also be modified through radiolabeling104, 105 for PET or SPECT as a

multimodal imaging probe. Some other magnetic NPs, such as metal alloys of FeCo

and FePt, Gd2O3,106 and MnO,107 are successfully utilized for in vitro cell labeling and

in vivo T1-weighted MRI.108, 109 Metallic gold nanostructures, generally NP, nanorod,

nanoshell, and nanocage, are efficient contrast agents in optical imaging based on


their unique SPR properties derived from the interaction of electromagnetic waves

with the electrons in the conduction band.110 This SPR enhances all linear and

nonlinear optical properties and thus offers multiple imaging modalities including

light scattering,111 extinction,112 two-photon luminescence,113 multiphoton

imaging,114 photothermal,115 and photoacoustic imaging.116 Efforts have been devoted

to the tuning of SPR imaging properties of Au NPs by morphological or surface

modifications117 and investigation of their surface interactions with cells.118, 119

1.4.3 Thermo-therapy agents

Magnetic NPs, mainly biocompatible SPION, have shown applications for cancer

treatment utilizing the locally generated heat, i.e. hyperthermia, to induce necrosis of

tumor cells using the energy absorbed from an oscillating magnetic field.120, 121

Clinical trials on feasibility of magnetic hyperthermia have been conducted for

patients with prostate cancers and a significant inhibition of prostate cancer growth

was found.122, 123 Other magnetic metallic NPs of Co, Fe or FeCo, with higher

magnetization compared to iron oxide, have been investigated for their hyperthermia

properties.124, 125

Another type of tumor treatment is by photodynamic therapy (PDT), which

involves cell destruction caused by toxic singlet oxygen or other free radicals that is

initiated by the reaction of a photosensitizer with tissue oxygen upon exposure to a

specific wavelength of light in the visible or NIR region.126 However, a major

drawback of PDT is that the photosensitizing drug stays in the body for a long time,

rendering the patient to be highly sensitive to light. An alternative to PDT is the

photothermal therapy (PTT) in which photothermal agents are employed to achieve

the selective heating of the local environment. Noble metal NPs, including gold

nanospheres,127 nanorods,111 nanoshells,128 and nanocages,129 are superior than

conventional photoabsorbing dyes as agents for PTT on account of their enhanced

absorption cross sections, which implies effective laser therapy at relatively lower

energies for minimal invasion, and higher photostability to avoid photobleaching. All

10

these types of plasmonic NPs can be prepared with their SPR peaks tuned to match

the center wavelength of laser irradiation for maximal absorbance, which should be

located in the NIR window (650–900nm)130 where light penetration is optimal due to

minimal absorption by water and hemoglobin in the tissue. Compared to other types

of nanoscale photothermal absorbers (e.g., carbon nanotubes131 and TiO2

nanotubes132), the plasmonic NPs have advantages on dual imaging/therapy functions

(see Section 1.4.2).

1.4.4 Stimuli-sensitive drug delivery agents

Nanomaterials that are sensitive to external stimuli are of emerging interest due to

their great potential in many biomedical and technical applications. For instance,

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

in which uptake and delivery of molecules can be controlled by a variety of external

stimuli, e.g. temperature,133, 134 pH value,43, 135 magnetic field,11 electric field,136

photoirradiation,18 ultrasound,19 biomolecules,20 and mechanical forces.137 The

previously discussed PDT is actually a stimuli-responsive system where the light of

specific wavelengths is used to trigger the therapeutic activity (see Section 1.4.3).

Among the environmentally sensitive nanomaterials, temperature-responsive

materials are probably one of the most commonly studied classes,138 and the idea

naturally came from the fact that many pathological areas demonstrate distinct

hyperthermia. Temperature-sensitive polymers of PNIPAAm and derivatives are the

most extensively used, which undergoes temperature-responsive conformational

change from the swelled, hydrophilic state to the shrunken, hydrophobic state when

above its LCST of 32-33 ˚C which is close to physiological temperature.87, 139, 140

Various kinds of PEGylated liposomes have been prepared for pH-sensitive drug

delivery,141, 142 which undergo degradation to release the contents upon exposure to

lowered pH in pathological sites such as tumors, infarcts, or inflammation zones.143-145


1.5 Biomedical applications of nanomaterials

1.5.1 Magnetic resonance imaging

1.5.1.1 Physical background

The basic principle of MRI is based on NMR which is the interaction between

magnetic field and protons. Under a given external magnetic field (B0, T), protons

experience a torque, since they cannot align exactly with the external field, which

make them precess around the direction of the field. The precessional frequency (ω0,

MHz) of the protons is found to be proportional to the external magnetic field, given

by the Larmor equation:

00 Bγω =

where γ is the gyromagnetic ratio (γ = 42.57 MHz T-1 for 1H), i.e. the ratio of the

magnetic dipole moment of a particle to its angular momentum. All the protons in a

magnetic field precess at the same Larmor frequency, which is a resonance condition.

Dependent on its internal energy, proton will precess in one of only two orientations,

either spin-up or spin-down.

When a resonant RF transverse pulse is perpendicularly applied to B0, it causes

resonant excitation of the magnetic moment precession into the perpendicular plane.

Upon removal of the RF, the magnetic moment gradually relaxes to equilibrium by

realigning to B0. Such relaxation processes involve two pathways:

a) longitudinal or T1 relaxation accompanying loss of energy from the excited

state to its surroundings (lattice) and recovery of decreased net magnetization (Mz) to

the initial state, and

b) transverse or T2 relaxation from the disappearance of induced magnetization on

the perpendicular plane (Mxy) by the dephasing of the spins.

These processes can be expressed as follows.

)1( 1Tt

z eMM−

−= (longitudinal)

12

2)sin( 0T

t

xy etMM−

Φ+= ω (transverse)

where T1 and T2 are the longitudinal and transverse relaxation time, respectively. Such

relaxation processes are recorded and then reconstructed by MRI to obtain gray scale

images.

1.5.1.2 Contrast agents

Biological tissues and organs have aqueous environments (70%-90% water in

most tissues) that vary in density and homogeneity, which are imaged as having

different contrast (signal) and provide anatomical information. Contrast agents help to

improve the specificity of MR imaging by producing an extra set of images with

different contrast, as a result of the interaction between the induced local magnetic

field and the neighboring water protons. The signal intensity of MRI primarily

depends on the local values of longitudinal (1/T1) or transverse (1/T2) relaxation rate

of water protons. The relaxivities of r1 and r2, which are commonly expressed in

mM-1 s-1, indicate respectively the increase in 1/T1 and 1/T2 per concentration [M] of

contrast agent:

][11

0,

MrTT i

ii

+= (i = 1, 2)

where Ti,0 is the relaxation times of native tissues (i.e., tissue devoid of exogenous

contrast agent). The efficiency of an MRI contrast agent is assessed in terms of the

ratio between the transverse and longitudinal relaxivities (expressed as r2/r1), which is

a defining parameter indicating whether the contrast agent can be employed as a

positive (T1) or negative (T2) agent. Commercially available T1 contrast agents are

usually paramagnetic complexes, e.g. Gd-DOTA (DotaremTM), Gd-DTPA-BMA

(OmniscanTM), and Mn-DPDP (TeslascanTM), while T2 contrast agents are based on

SPIONs, e.g. FeridexTM, ResovistTM, and CombidexTM. Due to some limitations of

these commercial contrast agents, such as polydispersity, poor crystallinity and

magnetic properties, new generation of contrast agents have being developed to

improve MR contrast effects with a less amount of dosage.


1.5.1.3 Superparamagnetism

An important requirement for useful T2 contrast agents is a large r2/r1 ratio, in

combination with high absolute value of r2. This condition can be achieved for agents

with predominantly outer sphere relaxation mechanism.146 Contrast agents that satisfy

these conditions are SPIONs. Another important parameter used to characterize ferri-

or ferromagnetic materials is anisotropy energy (Ea):

VKE aa =

where Ka is the anisotropy constant and V the volume of the crystal. Anisotropy

energy is the variation amplitude of magnetic energy of a nanomagnet dependent on

the direction of its magnetization vector.147 For the colloid of ferromagnetic crystals,

the return of the magnetization to equilibrium is determined by two different

processes, Néel relaxation and Brownian relaxation. When the magnetization curve is

perfectly reversible, because of thermodynamic equilibrium of system by fast

magnetic relaxation, the behavior has been named “superparamagnetism” by Bean

and Livingston.148 When the anisotropy energy of magnetic particles is larger than the

thermal energy kBT, where kB is the Boltzmann constant and T the absolute

temperature, i.e. TkEa B>> , the direction of magnetic moment maintains very close

to that of the anisotropy axes. In small crystals, the anisotropy energy is comparable

to the thermal energy, i.e. TkEa B≤ , so that the magnetic moment is no longer fixed

along the easy directions, which allows superparamagnetic relaxation. SPIONs can

cause noticeable shortening of T2 relaxation times with signal loss in the targeted

tissue and generate contrast to other tissues.

1.5.1.4 Preparation methods for magnetic NPs

Numerous chemical methods have been developed to synthesize magnetic NPs

for biomedical applications. There are some concerns for the synthetic methods

including development of a reproducible process without any complicated purification

14

procedure and experimental conditions leading to monodisperse magnetic grains with

suitable size.

Coprecipitation, the most widely used method, is probably the simplest and most

efficient chemical pathway to obtain magnetic NPs, where the soluble precursors

precipitate upon the addition of anionic counter ions. Iron oxides, either Fe3O4 or

γ-Fe2O3, can be prepared from aqueous Fe2+/Fe3+ salt solutions by the addition of a

base under inert atmosphere at room temperature or at elevated temperature.149 The

chemical reaction of Fe3O4 formation can be written as:

OHOFeOHFeFe 24332 482 +→++ −++

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

MnFe2O4,150 ZnFe2O4,151 and CoFe2O4.152 The size, shape, and composition of the

magnetic NPs are dependent on the type of salts used (e.g. chlorides, sulfates, nitrates),

the Fe2+/Fe3+ ratio, the reaction temperature, the pH value, and ionic strength of the

media. The advantages of coprecipitation method are high reproducibility of the NPs

and scalable yield, while the shortcoming is weak shape control and broad size

distribution. Efforts have been made in preparing monodisperse magnetite NPs.153

Thermo-decomposition method has been developed as an effect way to synthesize

high-quality semiconductor and oxide NPs with controlled size and shape,154, 155 in

which organometallic compounds as precursors are decomposed in high-boiling

non-aqueous media containing stabilizing surfactants.156, 157 The organometallic

precursors include metal acetylacetonates,154 metal-oleate complex,155 metal

cupferronates,158 or carbonyls.159 Fatty acids,160 oleic acid,161 and hexadecylamine162

are often used as surfactants. The separation of nucleation and aging period during the

particle formation allow the production of highly crystalline and monodisperse

magnetic NPs with selected particles sizes.

Other methods, including microemulsion,163 hydrothermal,164 and sol-gel,165 are

also investigated for synthesis of magnetic NPs. Several phase transfer strategies29, 166,

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

dispersion.


1.5.2 Plasmonic photothermal therapy

In photothermal ablation, optical irradiation is absorbed and transformed into heat,

inducing thermal denaturing of proteins and DNA in the cells, consequently causing

irreversible damage to the targeted tissue.168 It is desirable to use agents that are active

in the NIR region of the radiation spectrum to minimize the light extinction by

intrinsic chromophores in native tissue.130 Noble metal NPs have been used for the

application of photothermal cancer therapy,111 due to their strong electric fields at the

surface to enhance the absorption and scattering of electromagnetic radiation.

1.5.2.1 Surface plasmon resonance

Surface plasmons are surface electromagnetic waves that propagate in a direction

parallel to the metal/dielectric (or metal/vacuum) interface. The critical conditions for

existence of surface plasma are that the real part of the dielectric constant of the metal

must be negative and its magnitude must be greater than that of the dielectric, which

can be met in the IR-visible wavelength region for air/metal and water/metal

interfaces. The maximum and bandwidth of surface plasmon band are also influenced

by the particle shape, medium dielectric constant, and temperature. When plasmonic

NPs (gold or silver) are exposed to laser light resonant with their surface plasmon

oscillation, they can strongly absorb the light and rapidly convert it into heat via a

series of photophysical processes.169, 170

1.5.2.2 Preparation methods for gold NPs and NRs

In 1857, Faraday reported the formation of deep-red solutions of colloidal gold by

reduction of an aqueous solution of chloroaurate (AuCl4-) using phosphorus in CS2 (a

two-phase system) in a well-known work.171 Nowadays, various methods for the

preparation of gold colloids were reported and reviewed, after the breakthrough of

two classical works as citrate reduction method by Turkevitch172 and two-phase

method by Brust.173 Later, microemulsion,174 seeding growth,175 thermolysis176

16

methods were developed for synthesis of Au NPs. A popular method for growth of Au

nanorods is seed-mediated protocol originated by the work of Murphy et al.,177 in

which TSC-capped small Au NPs were added as seeds into growth solution containing

gold salt and surfactant for further growth of Au nanorods. Other methods include

earlier photochemical reduction,178 electrochemical,179 template,180 and top-down

lithographic methods.181

1.5.3 Temperature-triggered drug release

The driving force for the extensive studies on temperature-sensitive materials in

biomedicine is their potential for precise, on-demand content delivery in vitro or in

vivo. The controlled release of payload from a variety of carriers can be triggered by

illuminating with light at resonant wavelengths to enable photothermal conversion 182

or receiving thermal energy from the environment.37 Thermo-sensitive polymers are

one of the intensively studied materials for controlled drug release in combination

with proper drug carriers to form copolymers133 or core-shell structures.183

1.5.3.1 Thermo-responsive phase transition

PNIPAAm is a temperature-responsive polymer that was first synthesized in the

1950s.184, 185 In dilute aqueous solution, single chains of PNIPAAm undergoe a

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

LCST, where isolated, flexible but extended coil of polymer chain becomes collapse

and aggregation after the transition. When cross-linked, the hydrogel of PNIPAAm

shows a reversible LCST phase transition from a swollen hydrated state to a shrunken

dehydrated state, losing about 90% of its mass. Studies on gel transition theory show

that both hydrogen bonding and hydrophobic effect contribute to the driving force for

the transition, reflecting the changes in free energy.186, 187


1.5.3.2 Preparation methods for thermo-sensitive polymers

PNIPAAM has been synthesized mainly by the radical polymerization of

NIPAAm via a variety of techniques including the typical free radical initiation of

organic solutions188 and redox initiation in aqueous media.189 Other synthetic schemes

such as using ionic initiators190, 191 and radiation polymerization192, 193 have also been

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

RAFT133 and ATRP,194 have been developed to obtain controlled molecular weight

polymers with narrow polydispersity indexes and various complex architectures.133, 195,

196

18

2 Experimental

2.1 Magnetic mesoporous NPs

2.1.1 Synthesis of SPION and phase transfer

Monodisperse iron oxide NPs were synthesized using previously reported

method155 via thermal-decomposition of Fe-oleic complex in dioctyl ether at ca. 297

°C in the presence of oleic acid. Oleic acid capped Fe3O4 NPs were transferred to

water by mixing with aqueous CTAB solution at 65 ˚C and the emulsion was

vigorously stirred until all the chloroform was evaporated.

2.1.2 Fabrication of Fe3O4@mSiO2

The procedure of growing mSiO2 on CTAB capped Fe3O4 NPs is schematically

depicted in Figure 2.1. The aqueous Fe3O4 suspension was diluted with certain

amount of water and the pH was tuned to 12 by the addition of NaOH. The solution

was heated and, when the temperature of the solution reached 70˚C, a specific amount

of TEOS and corresponding four times of EtOAc were slowly added to the reaction

solution in sequence. After the reaction was conducted for 2 h, the core-shell particles

were collected by centrifugation and washed by EtOH two times. To extract CTAB

from the channels of mSiO2, the particles were dispersed in ethanolic NH4NO3

solution (10 mg/mL) at 60˚C and mixed for 30 min.197 After washing twice with

EtOH, the Fe3O4@mSiO2 NPs were re-dispersed in water for further use.

Figure 2.1 Synthetic diagrams of preparation of Fe3O4@mSiO2 core-shell NPs.


2.2 Preparation of thermo-sensitive composite NPs

The coating procedure of copolymer is schematically illustrated in Figure 2.2.

Prior to grafting of copolymer, Fe3O4@mSiO2 NPs without extraction of CTAB were

functionalized with carbon–carbon double bonds by reacting with TMSMA under

refluxing and N2 purging. The TMSMA-modified Fe3O4@mSiO2 NPs were used as

the seeds to conduct free radical copolymerization of NIPAAm and AAm on the NPs.

Typically, monomers of NIPAAm and AAm and cross-linker MBA were mixed with

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

flow before the initiator potassium persulfate (KPS) was introduced. The composite

NPs were then collected and washed by water to remove the unreacted monomers.

Figure 2.2 Synthesis procedure for preparation of core-shell composite Fe3O4@mSiO2@P(NIPAAm-co-AAm) NPs.

2.3 Synthesis of gold nanorods

In the present work, high AR gold nanorods were prepared by reducing the

growth solution of HAuCl4 and CTAB with the addition of HNO3 under ambient

temperature. A weak reducing agent, ascorbic acid, was used to reduce Au3+ to Au+

and a strong reducing agent of NaBH4 induced the formation of gold nanorods. Before

in vitro experiments, high AR gold nanorods were treated by a series of purification

steps to ensure the removal of CTAB from their surfaces but without causing

agglomeration. The procedure for synthesis of low AR gold nanorods is similar with

that for high AR ones with the only exception of using AgNO3 instead of HNO3 to

restrict the anisotropic growth of gold nanorods.198

20

2.4 Characterizations

The morphology of NPs was characterized by JEM-2100F field emission TEM

operating at an accelerating voltage of 200 kV. Powder XRD patterns were recorded

on a PANalytical XPert Pro powder diffractometer with Cu-Kα radiation (45 kV, 35

mA). Nitrogen sorption isotherms were obtained on a Micromeritics ASAP 2020 pore

analyzer at 77 K under continuous adsorption conditions. The BET equation was used

to calculate the surface area from adsorption data obtained in the range of relative

pressure p/p0 = 0.05 and 0.3. The total pore volume was calculated from the amount

of N2 adsorbed at p/p0 = 0.98. NLDFT model was applied to determine the PSD. The

hydrodynamic diameter of the particles was measured by PCS (DelsaTMNano particle

size analyzer, Beckman Coulter). The surface charge of NPs in suspension was

evaluated by Malvern Zetasizer. Concentrations of Fe or Au were measured by

Thermo Scientific iCAP 6500 series ICP-AES and Varian AAS, respectively. The

thermal behavior of composite NPs was measured DSC (Q2000, TA instruments). The

magnetization measurements were performed using a VSM (NUOVO MOLSPIN,

Newcastle Upon Tyne, Great Britain). The longitudinal NMRD profiles were recorded

at 37 °C on a Fast Field Cycling Relaxometer (Stelar, Italy) and the magnetic field

ranges from 0.013 MHz to 60 MHz. The radius of the magnetic particle r, which

determines the distance of closest approach between the paramagnetic center and the

water molecule, is determined by τD=r2/D from relaxometry data, where ωI is the

angular frequency of the proton precession, D is the relative diffusion coefficient

between the paramagnetic center and the water molecule. The specific magnetization

Ms relaxo can be obtained from Ms relaxo~(Rmax/CτD)1/2, where C is a constant and Rmax

the maximal relaxation rate in NMRD. Additional relaxivity r1 (20 and 60 MHz) was

measured at 0.47 T with a Minispec PC-20 Bruker spectrometer and 1.41 T with Mq

Series systems; r2 was measured with Minispec (20 and 60 MHz) on spectrometer

AMX-300.


2.5 Biocompatibility evaluation

2.5.1 Co-culture immature MDDCs with gold nanorods

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

were separated from buffy coats by standard Ficoll gradient centrifugation. MDDCs

were generated in the presence of IL-4 and GM-CSF as previously described.199 At

day 6 of culture, the immature MDDCs (4×105 cells/mL) were co-cultured with gold

nanorods at a concentration of 0.5, 5 or 50 µg/mL for 24 h. As controls, MDDCs

cultured in medium alone or with LPS (0.1 µg/mL; L8274, Escherichia coli, serotype

026-B6) were used. The morphology of MDDCs incubated with gold nanorods were

examined by Tecnai 10 TEM at 80 kV.

2.5.2 Flow cytometry analysis

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

were labeled with fluorescent phycoerythrin (PE) conjugated mouse monoclonal

antibodies (mAbs) specific for: CD1a and CD11c; and with fluorescein isothiocyanate

(FITC) conjugated mAbs specific for: CD40, CD80, CD83, CD86 and HLA-ABC,

HLA-DR and CD14 according to the manufacturer’s instructions. Control samples

were labeled with isotype-matched antibodies conjugated with the same fluorochrome.

Fluorescence was measured with a FACSCalibur flow cytometer. Ten thousand cells

were counted and analyzed by the program CellQuestPro.

2.5.3 Cell viability assay

To investigate the effect of gold nanorods on MDDCs’ viability, cells were

examined for the degree of apoptosis and necrosis by measuring the binding of

Annexin V-fluorescein and inclusion/exclusion of propidium iodide (PI) using a

FACSCalibur flow cytometer. PI-/Annexin V+ events defined early apoptosis whereas

PI+/Annexin V+ signals were counted as late apoptosis or secondary necrosis.

22

3 Results and discussions

3.1 Superparamagnetic mesoporous core-shell NPs

3.1.1 Morphology study

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

chloroform with narrow size distribution and an average diameter of 10.9 nm (σ ≈ 5.0

%). High resolution TEM image shows the single crystalline nature of the particles

(inset of Figure 3.1a). After phase transfer, aqueous SPIONs capped by CTAB

retained its morphology. Fe3O4@mSiO2 NPs with diameters varying from ca. 25 nm

to ca. 95 nm were synthesized. The low magnification images display that all the

particles are uniform and separated from each other (Figure 3.1). The insets of high

magnification images for all the core-shell NPs show a mSiO2 layer with about 2 nm

wormhole-like mesopores. We investigated the influence of reagents used for

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


synthesis of mSiO2 coatings and the optimized parameters for producing well-defined

core-shell structures are summarized in Table 3.1. Desired concentrations of CTAB

were chosen to assist phase transfer of hydrophobic magnetite NPs to aqueous phase,

and are responsible for the thickness of subsequent mSiO2 coating layer. For certain

amount of core materials, an excess amount of CTAB induces extra mSiO2 spheres

without SPION in the cores. On the other hand, deficiency of CTAB limits the shell

thickness despite the excessive amount of TEOS available. In the optimized system

with a specific ratio between Fe3O4, CTAB and water, where mSiO2 forms the coating

layer a single magnetite core, an excess amount of TEOS will not induce extra

core-free mSiO2 spheres, instead that TEOS condenses in the pores leading to pore

blockage. It is shown in Table 3.1 that, to grow thicker mSiO2 shell, the

[CTAB]/[Fe3O4] and [H2O]/[CTAB] have to increase to allow more TEOS to be

hydrolyzed and condenses around the magnetic core. The addition of EtOAc was

found to be crucial to produce well separated single-cored core-shell NPs.

Table 3.1 Summary of reactants composition for synthesis of Fe3O4@mSiO2 NPs with different particle sizes.

[CTAB]/[Fe3O4]a

Molar compositionb

Average particle

diameter, nmc 11.7 1 CTAB: 4.87 TEOS: 1.08 NaOH: 5983 H2O 25 11.7 1 CTAB: 9.74 TEOS: 2.15 NaOH: 11966 H2O 35 35.1 1 CTAB: 6.50 TEOS: 0.72 NaOH: 3989 H2O 45 35.1 1 CTAB: 9.74 TEOS: 1.44 NaOH: 7977 H2O 55 46.8 1 CTAB: 5.41 TEOS: 0.72 NaOH: 3989 H2O 65 70.2 1 CTAB: 8.12 TEOS: 1.08 NaOH: 5983 H2O 75 70.2 1 CTAB: 12.2 TEOS: 1.80 NaOH: 9972 H2O 85 93.6 1 CTAB: 12.2 TEOS: 2.02 NaOH: 11218 H2O 95

a 0.5 mL of Fe3O4 NPs suspension in CHCl3 (7.9 mg Fe/mL) was conducted for phase transfer; b CTAB refers to as the [CTAB] used previously for phase transfer; c particle diameters of Fe3O4@mSiO2 were determined by TEM (n=200).

24

3.1.2 Characterization of mesoporous silica

Small angle XRD pattern of Fe3O4@mSiO2 (Figure 3.2a) exhibits a characteristic

mesoporous structure with a reduction in the long-range mesoscale order indicated by

the absence of high order reflections.200 In Figure 3.2b, the N2 adsorption/desorption

isotherms exhibit a characteristic type IV isotherm as expected for mSiO2. The

corresponding pore size distribution (PSD) calculated by NLDFT model (inset of

Figure 3.2b) demonstrates a bimodal porosity (pore size 14 Å and 27 Å), which might

be induced by the heterogeneous distribution of magnetite NPs in the porous network

and hence a broad distribution of PSD. The BET surface area of 75 nm Fe3O4@mSiO2

NPs is 202 m2 g-1 and the total pore volume is 0.29 cm3 g-1.

Figure 3.2 Characterization of extracted Fe3O4@mSiO2 NPs dried at 80 ˚C, (a) small angle XRD pattern, (b) N2 adsorption (solid)-desorption (hollow) isotherms (inset: NLDFT slit type model for PSD from adsorption branch).

In wide angle XRD pattern, the broadest peak centered at 22˚ (2θ) (Figure 3.3b) is

consistent with the amorphous nature of the silica wall and the characteristic peaks of

a magnetite structure indicates that the magnetic core embedded in silica retains its

original crystalline nature after mSiO2 coating and surfactant extraction. The average

size of the Fe3O4 cores was calculated by using the Debye–Scherrer formula and is

determined as 10.5 nm, which is in good agreement with the average diameter

measured from TEM images, indicating they are dominantly single crystalline.


Figure 3.3 The powder X-ray diffraction (XRD) of (a) Fe3O4 and (b) Fe3O4@mSiO2.

3.1.3 Magnetometric and relaxometric properties

Field dependent magnetism of CATB-capped Fe3O4 and Fe3O4@mSiO2 NPs with

different shell thicknesses was examined at room temperature by using VSM. None of

the samples showed a hysteresis (Figure 3.4a), demonstrating that they are

superparamagnetic, which is a desirable characteristic for T2 MR contrast agents. The

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

are 48.7, 48.3, and 47.1 emu/g Fe, respectively. Since Ms determines the magnetic

moment and a higher value indicates a higher magnetic susceptibility, i.e. stronger

MRI signals,201 Fe3O4@mSiO2 NPs show implications of high MRI sensitivity. Fitting

the magnetization data to Langevin equation,148 we obtained the magnetic domain size

of the magnetic cores of these samples as 11.7, 11.8, and 11.8 nm respectively, which

Figure 3.4 (a) Field-dependent magnetization measurement of aqueous suspensions of CTAB-capped Fe3O4 and Fe3O4@mSiO2 (50 nm and 75 nm, repectively) NPs at room temperature, (b) NMRD profiles of CTAB-capped Fe3O4 and 50 nm Fe3O4@mSiO2 NPs recorded at 37 ˚C.

26

are close to the values obtained from TEM and XRD. On the other hand, the NMR

relaxometry plays an important role in evaluating the properties of the

superparamagnetic colloids as potential contrast agents.202 The effects of surface

coatings on the relaxometric properties of SPIONs are examined and CTAB-capped

and mSiO2-capped Fe3O4 NPs are compared through the characterization by NMRD

profiles (Figure 3.4b). By fitting the NMRD profiles with adequate theories,203, 204 the

average diameter of Fe3O4-CTAB is 12.5 nm and specific magnetization 48.2 emu g-1,

and τN 1.25×10-9 s, which are highly coherent with the results of magnetic domain

size and Ms by magnetometry. The NMRD of 50 nm-sized Fe3O4@mSiO2 provide the

diameter of the magnetite core embedded in mSiO2 as 28 nm, specific magnetization

as 14.3 emu g-1, and τN 5.85×10-9 s, which are much different from the results

obtained by magnetometry. This may be due to the effect of diffusive permeability of

Fe3O4@mSiO2 to water molecules, since the determination of magnetic crystal radius

r and specific magnetization Ms relaxo by NMRD are closely related with diffusion

parameters (e.g., τD and D).204, 205

3.1.4 MR studies of Fe3O4@mSiO2 NPs

The r1 and r2 relaxivity values and hydrodynamic sizes of Fe3O4-CTAB and

Fe3O4@mSiO2 with various coating thicknesses are summarized in Table 3.2.

Fe3O4-CTAB shows similar value of r2 with those of Fe3O4@mSiO2 NPs, however the

value of r1 is one magnitude higher than those of Fe3O4@mSiO2. Since the effects of

surface chemistry on relaxivity most likely arises from the hydrophilicity of the

coating layers and the coordination between the inner capping ligands, both

influencing spin orders,206 the variation of r1 is proposed to be due to the different

hierarchical structures between CTAB and mSiO2 layer around the magnetite core.

For Fe3O4@mSiO2 with varied coating thickness, it is found that the values of r1 are

effectively decreased by ca. 6 times (at 20 MHz) or ca. 4.5 times (at 60 MHz) when

particle size increases from 50 nm to 95 nm, whilst r2 is decreased by only ca. 40 % at


20 and 60 MHz. The magnitude of r1 is reported to be dependent on the magnetization

of the material, the electron spin relaxation time, and the accessibility of protons

bearing nuclear spins to the surface of iron oxide.207 The decreased value of r1 for

Fe3O4@mSiO2 with increased silica shell thickness may reflect the ability of mSiO2

coatings on separating water from the surface of the magnetite NPs, which is critical

for r1 values.208 Similar surface chemistry studies on relaxivity of polymer coated

Fe3O4 also showed that the hydrophilicity of the coating layer has a positive effect on

enhancing the proton relaxivities (both r1 and r2).167, 206 Superparamagnetic T2 contrast

agents were known to be able to produce a long-range magnetic field to promote the

spin-spin relaxation process of surrounding water molecules,23 and the magnitude of

r2 is believed to reflect the ability of the magnetic material to produce local

inhomogeneity in the magnetic field.209 Our results suggest that the locally generated

magnetic field by Fe3O4 cores has been weakened by the increased thickness of

mSiO2 coating.

Considering the NMRD data and hydrodynamic size measurement,

Fe3O4@mSiO2 NPs are assumed consisting of three regions: iron oxide core

generating a magnetic field, water impermeable layer with radius of rim, and slow

diffusion layer of protons with radius of rdiff (Scheme 3.1a). In Scheme 3.1b, thicker

Table 3.2 Mean hydrodynamic diameters and relaxivities (r1 and r2 are in unit of [s-1 mM-1]) of CTAB-capped Fe3O4 and different sized Fe3O4@mSiO2 NPs measured at 20 MHz (0.47 T) and 60 MHz (1.41 T) in water (37 °C), and the reported relaxivity values for commercial Feridex® and Resovist® contrast agents.

sample name mean 20 MHz 60 MHz

(particle diameter hydrodynamic

by TEM) diameter [nm]a

r1

r2

r2/r1

r1

r2

r2/r1

Fe3O4–CTAB (11 nm) 67 31.25 81.37 2.61 13.69 82.18 6.01Fe3O4@mSiO2 (50 nm) 60 3.65 84.26 23.1 1.31 92.13 70.3Fe3O4@mSiO2 (75 nm) 77 2.13 79.93 37.5 0.97 87.54 90.3Fe3O4@mSiO2 (95 nm) 96 0.61 50.13 82.2 0.31 55.44 179 Feridex208 72 40 160 4 − − − Resovist210 65 25 164 6.2 − − − a mean value of volume distribution

28

Scheme 3.1 Schematic representations of (a) a Fe3O4@mSiO2 NP labeled with rim (radius of water impermeable region indicated by dashed circle) and rdiff (radius of slow proton diffusion region indicated by dotted circle), (b) water diffusions in the coating zones with different thickness under magnetic field.

mSiO2 coated Fe3O4@mSiO2 has more water impermeable part than the thinner

coated one, therefore excludes more water and the relaxation rate of protons in

diffusion layer is slower, together resulting in the decrease of r2.

Owing to a higher impact of mSiO2 coatings on r1 than on r2, the r2/r1 ratio

increases as a function of the coating thickness, which are 82.2 and 179 at 20 MHz

and 60 MHz for 95 nm Fe3O4@mSiO2, respectively. These great enhancement of r2/r1

ratios, ca. 21 and ca. 14 times higher than the two commercial iron-oxide-based

contrast agents (see Table 3.2), indicates Fe3O4@mSiO2 a high efficiency on T2 MR

imaging. By comparison, in previous reports on relaxivity variation of conventional

amorphous SiO2 coated Fe3O4, there is no enhancement for r2/r1 ratios211, 212 when the

thickness of SiO2 coatings increases. The differences on the enhancement of r2/r1

ratios between conventional amorphous SiO2 and mSiO2 are most probably due to

their different properties on water permeability.

3.2 Multifunctional core-shell composite NPs

3.2.1 Morphological and structural studies

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

Fe3O4@mSiO2 NPs with different thicknesses of polymer layers. The thickness of

polymer coating was ca. 10 nm (Figure 3.5b) when 3 mmole of monomers were used,


Figure 3.5 TEM micrographs of (a) 85 nm Fe3O4@mSiO2 core–shell NPs, P(NIPAAm-co-AAm) coated 85 nm Fe3O4@mSiO2 composite NPs with different thicknesses of polymer layers using (b) 3 mmole, and (c) 12 mmole of NIPAAm and AAm monomers. The insets show the magnified images for each sample, and the polymer layer was visualized after positive stain with phosphotungstic acid

and it increased to ca. 30 nm (Figure 3.5c) when the amount of monomers increased

to 12 mmole. The observed necking of thick polymer coatings in Figure 3.5c is due to

a relatively high concentration of monomers and cross-linker. The thick polymer

coatings are reported to have less obvious hydrophilic–hydrophobic transition,

probably due to the existence of less flexible polymer chains.213 Hence, a thin layer of

polymer coatings is desired for thermo-responsive applications and to avoid blocking

the mesopores.

N2 adsorption/desorption isotherm (Figure 3.6a) of Fe3O4@mSiO2@P(NIPAAm-

co-AAm) NPs exhibit a characteristic type IV isotherm for mSiO2 structures. The pore

Figure 3.6 (a) N2 adsorption (solid)-desorption (hollow) isotherms, (b) PSD of extracted Fe3O4@mSiO2 (square) dried at 80 ˚C and Fe3O4@mSiO2

@P(NIPAAm-co-AAm) freeze dried at -78 ˚C (triangle).

30

size is calculated as 14 Å and 17 Å (a bimodal porosity). The BET surface area is 30.4

m2 g-1 and total pore volume is 0.15 cm3 g-1. A similar decrease of surface area and

pore volume after polymer grafting on mSiO2 was previously reported,134 where 19 %

decrease for heating-dried and 72 % decrease for freeze-dried thermo-sensitive

polymer coated NPs were observed.

3.2.2 Magnetic properties

Magnetic measurement of Fe3O4-CTAB, 85 nm Fe3O4@mSiO2, and

Fe3O4@mSiO2@P(NIPAAm-co-AAm) NPs was perform using VSM. None of the

samples showed a hysteresis (Figure 3.7), demonstrating that all of them are

superparamagnetic. The magnetic domain size of the magnetic cores of these samples

are obtained as 10.6, 10.4, 9.9 nm respectively according to Langevin equation.148

These values are comparable with the size estimated from TEM micrographs. The Ms

of CTAB-capped Fe3O4 is 57.2 emu g-1 Fe. For 85 nm Fe3O4@mSiO2 NPs, Ms is 52.7

emu g-1 Fe or 1.59 emu g-1 for Fe3O4@mSiO2 (ca. 3.0 wt % of Fe in Fe3O4@mSiO2).

The polymer-coated composite, prepared from a total of 3 mmol of NIPAAm and

AAm monomers (90%:10% in molar ratio), shows Ms of 49.1 emu g-1 Fe or 1.13 emu

g-1 for composite (ca. 2.3 wt % of Fe in composite). The T2-enhancing capability of

mSiO2 and polymer coated magnetite NPs is measured at 0.47 T, and r2 is obtained as

164 s-1 mM-1 and 113 s-1 mM-1, respectively.

Figure 3.7 Field-dependent magnetization measurement of Fe3O4 (■), Fe3O4@mSiO2

(○), and Fe3O4@mSiO2@P(NIPAAm-co-AAm) (△) at room temperature.


3.2.3 Thermo-responsive properties and manipulation of LCST

The colloidal properties of the Fe3O4@mSiO2@PNIPAAm NPs, prepared from 3

mmol of NIPAAm monomers, were studied by measuring the hydrodynamic particle

sizes. As shown in Figure 3.8a, the composite NPs maintain their initial size (ca. 200

nm, mean value of volume distribution) over a week, which indicates that they are

stable in phosphate buffered saline (PBS). The temperature-dependent hydrodynamic

size (Figure 3.8b) of the composite NPs were measured to show their

thermo-sensitivity. In Figure 3.8b, at 20 °C the particle size is 211 nm, and a sharp

decrease of the particle size is observed from 29 °C to 37 °C due to the collapse of the

PNIPAAm chain. The particle size is 144 nm at 37 °C and almost no change is

observed till 45 °C. By fitting the variation of hydrodynamic size on temperature

change, phase transition temperature, i.e. LCST, of the polymer coated composite NPs

is found at ca. 33 °C.

The thermo-responsive properties of the Fe3O4@mSiO2@P(NIPAAm-co-AAm)

NPs and the LCST of the polymer shell were further studied utilizing DSC. An

aqueous suspension of PNIPAAm coated Fe3O4@mSiO2 NPs using 1.5 mmol

NIPAAm was observed to exhibit an endothermic peak at 36.9 ˚C in heating process

and an exothermic peak at 34.7 ˚C in cooling process (Figure 3.9a). Such hysteresis of

phase transition may be due to kinetic effects. When the amount of monomer of

NIPAAm increases to 3 mmol, a similar endothermic peak of polymer coated NPs was

Figure 3.8 Hydrodynamic diameters (average mean values of volume distribution ± standard deviation) of Fe3O4@mSiO2@PNIPAAm NPs in PBS (a) at 25 °C for different temporal points, (b) versus temperature

32

Figure 3.9 DSC curves of Fe3O4@mSiO2@PNIPAAm samples show phase transitions and the variation of transition temperatures with the increase of monomer amount from (a) 1.5 mmol to (b) 3 mmol of NIPAAm in three or five continuous heating–cooling cycles, respectively. found at 34.8 ˚C and an exothermic peak at 32.8 ˚C (Figure 3.9b). Both of the

polymer coatings show a good reversibility of the phase transition demonstrated by

three or five continuous heating–cooling cycles, respectively. When the cross-linking

density is fixed, i.e. 10 % weight percent of MBA in monomers, LCST is decreased

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

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

LCST becomes lower.214 Compared with the LCST determined by DLS as 33 °C, the

lag of response in DSC measurement showing endothermic peak at 34.8 ˚C might be

due to the slow equilibration of the temperature in the measuring cell.

On the other hand, LCST can also be manipulated by adjusting the ratio of

hydrophobic and hydrophilic segment of the polymer. In the present work, we

introduce a hydrophilic co-monomer of AAm to produce copolymer with NIPAAm. A

total of 3 mmole of NIPAAm and AAm with varied molar ratios were applied to coat

the same amount of Fe3O4@mSiO2 NPs as PNIPAAm coated ones with the

cross-linking density unchanged. Figure 3.10 shows that the endothermic peaks of 5

%, 10 %, and 15 % AAm content of the co-polymer coatings are located at 37.6 ˚C,

40.1 ˚C, and 42.4 ˚C, respectively, while the exothermic peaks are at 35.3 ˚C, 37.6 ˚C,

and 39.6 ˚C, respectively. All samples with different compositions showed reversible

phase transition behavior in five continuous heating-cooling cycles. We found that


Figure 3.10 DSC curves of Fe3O4@mSiO2@P(NIPAAm-co-AAm) samples show phase transitions and the variation of transition temperatures with the increase of co-monomer AAm percentage as (a) 5%, (b) 10%, and (c) 15% in total 3 mmol of NIPAAm and AAm monomers. All samples were conducted for five continuous heating–cooling cycles. more AAm content displays a higher LCST than those with less AAm content, which

is contrary to the observation for PNIPAAm polymer shell. These results also suggest

that LCST can be accurately controlled to shift for a few degrees, which is ca. 2.0–2.5

˚C increase per 0.15 mmol AAm addition in the present work. For in vivo applications,

the LCST should be tuned to a value above the body temperature (37 ˚C) but below

the hyperthermia temperature (42 ˚C).215 Our system with subtle LCST tuning

properties indicates the possibility to manipulate the drug release temperature for such

application.

Figure 3.11a shows that Fe3O4@mSiO2@P(NIPAAm-co-AAm) NPs have a very

weak interaction with external magnetic field (300 mT) at room temperature and can

be stable for few hours, which is usually seen for superparamagnetic NPs with

diameter smaller than 20 nm. It is obviously not suitable for magnetic separation

Figure 3.11 Photographs of Fe3O4@mSiO2@P(NIPAAm-co-AAm) aqueous suspension under the influence of a magnetic field (300 mT) at (a) 20 ˚C and (b) 40 ˚C.

34

applications. Currently, large magnetic particles or beads usually with diameters of

few micrometers are applied for magnetic separation because of their rapid and strong

response to external magnetic field. However, their low surface-to-volume ratio and

large hydrodynamic size limit their conjugation efficiency and circulation time, i.e.

blood half life. Figure 3.11b shows that the thermo-sensitive Fe3O4@mSiO2@

P(NIPAAm-co-AAm) NPs can respond to an external magnetic field rapidly when the

temperature is above their LCST because of their hydrophobic surface and forming

aggregation, similar with the previous reported PNIPAAm coated magnetic

microspheres.216 This behavior is reversible over several cycles and the polymer

coated NPs became soluble again when temperature is below LCST.

3.3 High aspect ratio gold nanorods

3.3.1 Morphological and structural studies

The effects of different concentrations of HNO3 (0.2-72 mM) on the morphology

of gold nanorods have been studied. Parts of the TEM micrographs are presented in

Figure 3.12. The length and AR of gold nanorods were found to be greatly influenced

by the HNO3 concentration, and the average length of gold nanorods varied from ~21

to 447 nm and the AR changed from ~1 to 26. The highest AR was achieved using 3

mM HNO3, while a further increase of HNO3 concentration resulted in the formation

of shorter nanorods, and finally spherical NPs were obtained at a concentration of 72

mM HNO3 (Figure 3.12d).

Figure 3.12 TEM images of gold nanorods prepared by using HNO3 with the following concentrations: (a) 0, (b) 1 mM, (c) 3 mM, and (d) 72 mM in the solution.


Figure 3.13 (a) TEM low resolution image of an individual gold nanorods prepared with 1 mM HNO3 and the SAED pattern (inset); (b) HRTEM images showing lattice fringes at the edge of the gold nanorods; (c) XRD pattern of high AR gold nanorods prepared with 1 mM HNO3 and deposited onto a glass slide.

The crystal structure of gold nanorods was studied by HRTEM on a

representative sample prepared in the presence of 1 mM HNO3 solution. The

micrograph (Figure 3.13a) shows typical faceted ends of nanorods. The selected area

electron diffraction (SAED) pattern of this nanorod (inset; Figure 3.13a) exhibits

additional reflections indicating twinning. An HRTEM image of the end region of the

gold nanorod (Figure 3.13b) shows lattice fringes with a d-spacing of 2.35 Å, which is

assigned to Au (111) planes. Gold nanorods were also examined by XRD (Figure

3.13c) where a very strong (111) diffraction peak was observed, which indicates that

the evaluation from the HRTEM image of a single nanorod is valid for the whole

ensemble.

3.3.2 Particle growth mechanism studies

An attempt to elucidate the mechanism of formation of high AR gold nanorods

was carried out by studying the morphology and crystal structure of gold nanorods

formed at different growth stages through HRTEM. Figure 3.14a (inset) shows images

of gold NPs (AR ~1.2) which were formed 5 min after the introduction of the

reducing agent in the presence of 1 mM HNO3. Most of the formed particles were

found to be twinned, indicated by the crystal lattices pointing to different directions

but with same lattice spacing (Figure 3.14a). These gold NPs grew anisotropically

36

Figure 3.14 TEM images of gold NPs synthesized in the presence of 1 mM HNO3 showing crystal lattice and morphology (inset) at a growth duration of (a) 5 min, (b) 1 h; and (c) Fourier domain image of the particle in (a) by FFT process shows three pairs of spots with the same radial distance (indicated by arrows) that corresponds to the three marked lattice fringes in (a). All scale bars in white are 5 nm. into rod-shaped particles with ARs of ~17 after 1 h (Figure 3.14b). Fast Fourier

transform (FFT) analysis of Figure 3.14a exhibits several pairs of spots (indexed in

the Figure 3.14c) with the same radial distance from the center spot, indicating that

these spots represent the same crystal lattice plane and that their mutual orientation is

due to twinning. Exploration by inverse FFT (see Supplementary material in

corresponded paper) shows that the lattice spacing of these five-fold twinned gold

NPs is of ca. 2.4 Å, close to that of the Au (111) lattice plane.

Further examination of the effect of nitric acid on the growth of gold NPs (Figure

3.15) shows that not only the morphology but also the crystal structure of gold NPs

was greatly influenced by the concentration of HNO3. For the nanorods produced with

72 mM HNO3, only single-crystal spherical NPs were formed at the initial stage,

different from the twinned crystal structure when using 1 mM HNO3. Furthermore,

these NPs grew isotropically, instead of anisotropically, with their diameter increasing

from ~7 to ~15 nm within 1 h (insets of Figure 3.15a-b). Under these conditions,

about 6% (12 out of 200) of the NPs showed five-fold twinned decahedral shape

(Figure 3.15b), while they did not grow into rod-shaped particles. The HRTEM image

of the initially formed NPs (Figure 3.15a) shows only one set of clearly resolved

lattice fringes (indicated by arrows) and the pair of spots (with radial symmetry) in

Fourier domain image (Figure 3.15c) corresponds to that lattice fringes. Inverse FFT

analysis shows that the lattice spacing is ~2.0 Å, which matches with an Au (200)


Figure 3.15 TEM images of gold NPs synthesized in the presence of 72 mM HNO3 showing crystal lattice and morphology (inset) at a growth durations of (a) 5 min, and (b) an example of few particles with decahedron structures after 1 h; (c) Fourier domain image of the particle in (a) (the one with the lattice plane indicated) by FFT process. The pair of the most intense spots (with radial symmetry), which is indicated by an arrow, corresponds to the most intense lattice fringes in the HRTEM image. All scale bars in white are 5 nm. lattice plane and confirms that these single-crystal gold NPs were grown on {100}

lattice planes (see Supplementary material in corresponded paper).

We hypothesize that the strong oxidative dissolution of Au(0) seeds at high

concentration of HNO3 in the presence of chlorine ions is responsible for no existence

of twin defects at the earlier stage of NP growth, which are essential for the

anisotropic growth of NPs into rods.217 It is noticed that HNO3 has a lower reduction

potential than HAuCl4, so its oxidative property can only be effective at high

concentration. Conversely, multiple-twinned gold NPs were formed intermediately

and grew into elongated nanorods at low HNO3 concentration, which implies that the

effect of nitrate ions on elongation of the surfactant micelles218 is predominant over

the oxidative dissolution of multiple twinned particles, the former effect being

responsible for growth of long gold nanorods. On the other hand, the oxidative

dissolution by HNO3 would assist the molecules on the surface of a small

(energetically unstable) particle to diffuse and then deposit onto unbound (111) facets

of larger particles to lower the overall energy of the system, i.e., Ostwald ripening.219

Therefore, the mechanism of growth of high AR gold nanorods can be attributed to

the effect on elongation of surfactant micelles by nitrate ions and the process of

Ostwald ripening. Consequently, there is only a narrow window of HNO3

concentration that results in high AR gold nanorods.

38

3.3.3 Biocompatibility evaluation of high AR gold nanorod

3.3.3.1 Viability studies

MDDCs cultured in medium alone were used as a negative control, cells treated

with LPS (0.1 µg/mL) as a positive control, while spherical gold NPs were used as a

reference.199 It is found that nanorods with high AR seem not to have an impact on

cell viability (Figure 3.16a, b) and similar results were seen for the spherical gold NPs.

In contrast, nanorods with low AR at a concentration of 50 µg/mL induced a

considerable decrease in viability from 84 ± 6.6 % (medium control) to 57 ± 18 %

(Figure 3.16a), involving mainly late apoptosis (programmed cell death) and necrosis

(uncontrolled cell death) (Figure 3.16b). At the two lower concentrations of similar

type of nanorods, no increased cell death was observed (Figure 3.16a) compared to

the medium control.

To elucidate the different effects seen for the two rod types, the chemicals used

during the two production processes were investigated in the same viability analysis

as above. The amounts of chemicals used during the particle preparations were added

to MDDCs without nanorods, as well as ten fold lower amounts, to simulate the

dilution caused during the particle washing steps. AgNO3 was only used for the low

AR rods and HNO3 for the high AR rods. However, neither of these precursors nor

L-ascorbic acid seems to have any considerable effects on viability (Figure 3.16c, d).

CTAB did so and is concentration dependent.

In a second set of experiments, we used a larger number of washing steps of the

rods to reduce the amount of CTAB bound on gold nanorods. Hereby improved

biocompatibility was achieved for the low AR rods, but it did also cause particle

agglomeration. This suggests that the observed decrease of viability induced by the

low AR rods were not due to different morphology, but rather due to higher

concentrations of CTAB, which could not be efficiently washed away compared to the

case of high AR rods. We propose that the addition of HNO3, used for synthesis of

high AR rods, changes the equilibrium of the chemical reaction in the colloidal


Figure 3.16 Flow cytometry data showing percentage viable cells (a, c), and percentage early, and late apoptotic and necrotic cells (b, d) after 24 h co-culture with either NPs (a, b) or with chemicals used for the preparations of high (~21) and low AR (~4.5) gold nanorods (c, d) Results represent the mean ± SEM (standard error of the mean) from 3 (a, b) or 4 (c, d) experiments, respectively, using cells from different healthy blood donors. MDDCs cultured in medium alone were included as a negative control and LPS was included as a positive control.

suspension, thereby facilitating the removal of CTAB molecules from the surface of

gold nanorods. The results from our MDDC cultures show that this is important for

their higher biocompatibility.

3.3.3.2 Immune modulatory effects

The biocompatibility of the high AR gold nanorods were further evaluated in

terms of immune modulatory effects on MDDCs, after co-culture for 24 h. Results

showed that the rods did not have any impact on the overall expression of CD40,

CD80, CD83 or CD86 on MDDCs as measured by mean fluorescence intensity

40

Figure 3.17 Expression of the MDDC surface molecules CD40, CD80, CD83 and CD86 were measured by flow cytometry after 24 h co-culture with spherical gold NPs or high AR (~21) gold nanorods and are expressed as mean fluorescence intensity (MFI). For each sample, 104 cells within the gate for viable MDDCs were analyzed. LPS was used as a positive control. Results represent the mean ± SEM from 3 independent experiments using cells from different healthy blood donors.

(Figure 3.17). However, the percentage of CD86+ cells was slightly increased in a

dose dependent manner, as seen in Figure 3.18, which confirms our previous study on

spherical gold NPs.199 We can therefore conclude that neither the high AR nanorods

nor the spherical NPs induced any pronounced maturation of the MDDCs, thus

different morphologies of gold NPs did not seem to cause any different cellular

responses.

Figure 3.18 Expression of the MDDC surface molecules CD40, CD80, CD83 and CD86 were analyzed by flow cytometry after 24 h and are expressed as percentage of positive cells. For each sample, 104 cells within the gate for viable MDDCs were analyzed. LPS was used as a positive control. Results represent the mean ± SEM from 3 independent experiments using cells from different healthy blood donors.


3.3.3.3 Cellular internalization study

To explore the interaction between high AR rods and MDDCs, we also

investigated whether the gold nanorods are internalized by MDDC using TEM.

Within 1 h of co-incubation with nanorods (50 µg/mL), it could be detected that these

nanorods were inside the cells and packed within vesicular structures, probably

endolysosomes.220 After 24 h most cells contained high amounts of nanorods (Figure

3.19a, b) with a similar uptake in vesicular structures as at 1 h, demonstrating an

efficient uptake by MDDCs. These observations were also seen with the spherical

gold NPs. The relationship between the surface coating of gold nanorods and cellular

uptake has been studied previously, and it was found that the binding of serum

proteins plays a significant role in nanorods uptake.221 However, the MDDC uptake of

the gold nanorods analyzed in this study requires further investigation.

Figure 3.19 TEM images of MDDCs show internalized high AR (~21) gold nanorods after 24 h (a), which reside within vesicular structures, as visualized by increased magnification (b).

42

4 Conclusions

In this thesis, multifunctional nanomaterials have been developed through various

methodologies for biomedical applications, such as contrast agents for MRI and

thermo-sensitive magnetic separation.

The mSiO2 is coated on a single core of Fe3O4 NP to form a uniform core-shell

structure with tunable coating thickness. We have investigated parameters responsible

for producing high quality core-shell structures. Relaxivity studies of these core-shell

NPs showed better contrast properties with enhanced r2/r1 ratio as a function of the

coating thickness, which is 20 fold higher than that of the commercial contrast agents.

We ascribe the mechanism of such enhancement to the effect of mSiO2 coating layers

on water exclusion and diffusion.

A multifunctional nanomaterial has been developed by grafting a

thermo-sensitive copolymer P(NIPAAm-co-AAm) as the outer shell onto

Fe3O4-mSiO2 NPs. In addition to the superparamagnetic property, high r2 relaxivity,

and large surface area, we found that the phase transition temperature of the core-shell

composite can be finely adjusted from ca. 34 to ca. 42 ˚C by tuning the quantity or

composition of the co-monomers. The integrated capabilities from different

components of the multifunctional NPs make these materials a novel candidate for

future medical diagnosis and therapy.

A new synthesis method of gold nanorods with high AR has been developed

without using pre-made seeds of gold NPs. The growth mechanism of high AR gold

nanorods has been explained by the effects of HNO3 on elongation of micelles and

oxidative dissolution of Au(0) together with Ostwald ripening. The biocompatibility

of the nanomaterials is evaluated by studying the immune modulatory effects with

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

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

activation, even though the particles were efficiently internalized by the cells, which

exhibit a high potential of this material for medical applications.


Future work

A few designs for construction of multifunctional NPs can be developed for

several biomedical applications, namely thermo-therapy, dual-modality MRI, and

drug release.

Specifically, one possible hierarchical structure may be composed by growing Au

nanorods in the mesoporous channels of Fe3O4@mSiO2 NPs to enable both magnetic

imaging and NIR-induced local heating simultaneously for the improvement of

medical diagnosis and therapy. Certain surface functionalization of mSiO2 will

facilitate the realization of other applications, such as cancer cell targeting by

conjugating with folic acid through aminopropyltriethoxysilane (APTMS), magnetic

separation or targeting using thermo-sensitive polymer.

A proper design on construction of assembly of different magnetic materials

could be useful for dual modal MRI, where two different T1 and T2 imaging modes are

achieved simultaneously, potentially providing highly accurate information. For such

purpose, typical T1 contrast agents Gd-chelate or amorphous Gd2O(CO3)2 can be

attached or formed in situ on the surface of Fe3O4@mSiO2. This system may be

potentially capable for the imaging of a wide range of biological targets with

enhanced diagnostic accuracy.

For controlled drug release, one possible design incorporates the synthesis of a

copolymer of thermal sensitive polymers (e.g. PNIPAAm) and biodegradable

polymers (e.g. PLLA, PDLA, PLGA) as drug carriers. Their hydrophobic interior can

be loaded with water-insoluble anticancer drug and photosensitive agent (e.g. initially

hydrophilic Au nanorods phase transferred to non-polar solvent) for thermo-triggered

drug release.

44

Acknowledgements

First of all, I would like to thank my supervisor, Prof. Mamoun Muhammed, for

giving me the precious opportunity to join the Functional Materials Division and

leading me to the world of nanotechnology. His enlightening ideas and kindness are

always strong supports during my study.

I would like to thank Assoc. Prof. Muhammet Toprak for his great help from

research to everyday life, especially for his kind help in revising my manuscripts. I

wish him a great success in the scientific career.

I want to thank Dr. Jian Qin for his great patience, scientific discussion and

professional guidance on my first step of research. Thanks to Dr. Shanghua Li and Dr.

Andrea Fornara for the help, encouragement, and invaluable advices. I wish all of

them a splendid future for their career either in ABB or YKI, as well for the family. I

am very grateful to Abhi for the thorough linguistic check of the thesis. Many thanks

to the members in Functional Materials Division, Dr. Salam, Ying, Xiaodi, Sverker,

Carmen, Robina, Mazher, Terrance, Mohsin, Nader, Yichen, Dr. Mohammed and all

former colleagues, for their friendship and support. I appreciate Hans and Wubshet for

their great work of maintaining and helping on use of electron microscopes.

I am grateful to my coworkers from Karolinska Institute for the fruitful

collaborations: Dr. Helen Vallhov, Dr. Britta Andersson, Assoc. Prof. Susanne

Gabrielsson and Prof. Annika Scheynius from Department of Medicine; Neus Feliu

and Prof. Bengt Fadeel from Institute of Environmental Medicine. I would also like to

thank Dr. Sophie Laurent, Dr. Alain Roch and Prof. Robert N. Muller from University

of Mons in Belgium for the productive collaboration and great help on MR

experiments. Many thanks to Lin Dong and Assoc. Prof. Sergei Popov from Optics

Division, KTH, for the great collaboration.

Last but not least, I would like to thank my parents for their endless support and

encouragement, and the most important person, my wife Min Tian, for her deep love

and a strong support for my scientific career.


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