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«The Dissertation Committee for Li Ma Certifies that this is the approved version of the following dissertation: Textured Thin Metal Shells on Metal ...»

-- [ Page 1 ] --

Copyright

by

Li Ma

The Dissertation Committee for Li Ma Certifies that this is the approved version of

the following dissertation:

Textured Thin Metal Shells on Metal Oxide Nanoparticles with Strong

NIR Absorbance and High Magnetization for Imaging and Therapy

Committee:

Keith P. Johnston, Supervisor

Marc D. Feldman, Co-Supervisor

Thomas E. Milner

Konstantin V. Sokolov

Charles B. Mullins

Gyeong S. Hwang

Textured Thin Metal Shells on Metal Oxide Nanoparticles with Strong NIR Absorbance and High Magnetization for Imaging and Therapy by Li Ma, B.E.; M.E.; M.A.

Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin December 2010 Dedication To my wife, son, parents and friends Acknowledgements A dissertation for the degree of Doctor of Philosophy is a valuable treasure for human being. The rule of treasure is equal to everybody. You can’t take it with you when you leave. Some people think treasure equals to money, others think the real treasure is human being herself. When I look back to see what I have done for this dissertation, I really enjoy the procedure of digging these treasures out of the ground. The procedure of learning is the only important thing to achieve treasures and the degrees. I guess that is why we call this dissertation for Doctor of Philosophy, not Doctor of Chemical Engineering. I am lucky to be able to find these treasures earlier in my life. And I want to share them with you in this dissertation.

I sincerely thank all people who help me discovered the treasures and achieved the goal for my future career. They are my wife Xi Ke, my son Yuntao Ma and my parents who are always patient to support me for every choice I have made. They are my advisor Keith Johnston, who worked with me closely in the past 5.5 years and other advisors or sponsors: Marc Feldman, Thomas Milner, Konstantin Sokolov and Stanislav Emelianov who contributed a lot of time and helpful discussions with me. They are my people, who helped me in the treasure discovery procedure: Ameya Borwankar, Justina Tam, Jasmine Tam, Brian Willsey, Daniel Rigdon, Vidia Paramita, Davis Ingram, Tianyi Wang, Amit Paranjape, Joseph Villard, Kiran Cheruku, Timothy Larson, Travis Jenkins, Min Qu and Mohammad Mehrmohammadi. They are my lab mates, who always sit behind me to support: Andrea Miller, Avinash Murthy, Aileen Dinin, Dan Slanac, Mehul Patel, Xi Chen and Yunsheng Chen.

–  –  –

The ability of sub 100 nm nanoparticles to target and modulate the biology of cells will enable major advancements in cellular imaging and therapy in cancer and atherosclerosis. A key challenge is to load an extremely high degree of targeting, imaging, and therapeutic functionality into small, yet stable particles. A general mechanism is presented for thin autocatalytic growth on nanoparticle substrates (TAGS), as demonstrated for a homologous series of 5 nm textured Au coatings on 42 nm iron oxide cluster cores. Very low Au supersaturation levels are utilized to prevent commonly encountered excessive autocatalytic growth that otherwise produce thick shells. The degree of separation of nucleation to form the seeds from growth is utilized to control the morphology and uniformity of the thin Au coatings. The thin and asymmetric Au shells produce strong near infrared (NIR) absorbance with a cross section of ~10-14 m2, whereas the high magnetic content per particles provides strong r2 spin-spin magnetic relaxivity of 200 mM-1s-1. TAGS may be generalized to a wide variety of

–  –  –

variety of applications as diverse as catalysis and bionanotechnology.

High uptake of the nanoclusters by macrophages is facilitated by the dextran coating, producing intense NIR contrast both in cell culture and an in vivo rabbit model of atherosclerosis. A novel conjugation technique further allows covalent binding of anti-epidermal growth factor receptor (EGFR) monoclonal antibody (Ab) to the nanoclusters for highly selective targeting to EGFR over expressing cancer cells.

AlexaFluor 488 tagged Ab nanocluster conjugates were prepared to correlate the number of conjugated Abs with the hydrodynamic diameter. The high targeting efficacy was evaluated by dark field reflectance imaging and atomic absorbance spectrometry (AAS).

Colocalization of the nanoparticles by dual mode in-vitro imaging with dark field and fluorescence microscopy demonstrates the Abs remained attached to the Au surfaces.

The extremely high curvature of the Au shells with features below 5 nm influence the spacing and orientations of the Abs on the surface, which has the potential to have a marked effect on biological pathways within cells. These targeted small multifunctional nanoclusters may solve some key molecular imaging challenges for cancer and atherosclerosis.

–  –  –

List of Figures

Chapter 1 Introduction

1.1 Application of multifunctional nanoclusters

1.2 Objectives

1.2.1 Control the size and shape of nanoroses

1.2.2 Synthesis antibody conjugated nanorose for targeted delivery.....5





1.3 Dissertation outline

1.4 References

Chapter 2 Small Multifunctional Nanoclusters (Nanoroses) for Targeted Cellular Imaging and Therapy

2.1 Introduction

2.2 Results and discussion

2.2.1 Synthesis, structure and stability

2.2.2 Optical and magnetic properties in solution

2.2.3 Cell targeting, imaging and photothermolysis

2.2.4 Macrophage cell uptake specificity

2.2.5 Atherosclerotic rabbit aorta imaging

2.3 Conclustions

2.4 Methods

2.4.1 Synthesis of iron oxide dispersion

2.4.2 Nanorose growth and purification

2.4.3 Dynamic light scattering

2.4.4 Determination of absorption and scattering with an integrating sphere

2.4.5 In vitro magnetic resonance imaging

2.4.6 In vitro macrophage photothermolysis study

2.4.7 Hyperspectral microscopy

viii 2.4.

8 Atherosclerotic rabbit model

2.5 Supporting information

2.5.1 Reproducibility in particle size distribution

2.5.2 Porosity of dextran in the shells about the iron oxide particles..45 2.5.3 Estimation of number of particles per nanocluster

2.5.4 Rabbit endothelial cell preparation

2.4.5 Rabbit Smooth Muscle Cell Preparation

2.6 References

Chapter 3 Growth of Textured Thin Au Shells on Iron Oxide Nanoparticles...59

3.1 Introduction

3.2 Experimental section

3.2.1 Materials

3.2.2 Kinetics of nucleation and growth of Au on iron oxide..............63 3.2.3 Coating Au on iron oxide nanoclusters with addition of precursor in iterations or continuously

3.2.4 Materials characterization

3.3 Results

3.3.1 Au nucleation and growth kinetics on iron oxide substrates with a single iteration

3.3.2 Au nucleation and growth kinetics on iron oxide substrates with multiple iterations

3.3.3 Au nucleation and growth kinetics on iron oxide substrates with continuous precursor addition

3.3.4 The separation of Au coated iron oxide nanoparticles from uncoated ones by centrifugation

3.3.5 Magnetic properties of the Au coated iron oxide nanoparticles.81

3.4 Dissussion

3.4.1 Autocatalytic nucleation and growth of pure Au nanoparticles..83 3.4.2 Heterogeneous nucleation and growth of Au shells on substrates for a single iteration at a relatively low Au3+/Fe ratio

3.4.3 Heterogeneous nucleation and growth of Au shells on substrates with iterative or continuous Au3+ addition

ix 3.4.4 Passivation and steric stabilization with mPEG-thiol.................88 3.4.5 SPR Spectra for various particle morphologies

3.5 Conclustions

3.6 Supporting information

3.6.1 Iron oxide nanocluster synthesis

3.6.2 The estimation of gold shell thickness on the iron oxide nanoclusters

3.6.3 The estimation of centrifugation speed for particle sedimentation

3.6.4 The thermodynamic driving force for reduction of Au3+ on gold surfaces versus homogeneous reduction in solution

3.7 References

Chapter 4 Tuning the Nucleation and Growth of Au Shells on Iron Oxide Nanoclusters

4.1 Introduction

4.2 Experimental section

4.2.1 Materials

4.2.2 Coating Au on iron oxide nanoclusters

4.2.3 Materials characterization

4.3 Results

4.3.1 Effect of pH on kinetics of formation of pure Au nanoparticles113 4.3.2 Effect of pH on nucleation and growth of Au coatings on iron oxide at high polymer concentration

4.3.3 Calculation of size distribution moments

4.3.4 Effect of mPEG-SH concentrations on the morphology...........120

4.4 Discussion

4.4.1 Homogeneous nucleation and autocatalytic growth to form pure metal nanoparticles

4.4.2 Mechanism of nucleation and growth of thin Au films on iron oxide substrates

4.4.3 Effect of polymer: passivation and steric stabilization.............127 4.4.4 Homologous series of shell morphologies by TAGS...............128 x 4.4.5 SPR behavior

4.5 Conclustions

4.6 Supporting information

4.7 References

Chapter 5 Selective Targeting of Antibody Conjugated Multifunctional Nanoclusters (Nanoroses) to Epidermal Growth Factor Receptors in Cancer Cells.......143

5.1 Introduction

5.2 Experimental section

5.2.1 Materials

5.2.2 Antibody conjugation to nanorose

5.2.3 Cell culture and nanoparticle targeting

5.2.4 Au elemental analysis in the labeled cells

5.2.5 Hydrodynamic diameter measurement of Abs conjugated nanoparticles

5.2.6 Fluorescent labeling and UV-vis spectrometry

5.2.7 Dark field reflectance imaging and fluorescent imaging..........152

5.3 Results

5.3.1 Number of clone 225 Abs conjugated to nanorose and hydrodynamic diameter

5.3.2 Selective cellular uptake with dosage response

5.3.3 Low cell uptake in EGFR(-) control cancer cells

5.3.4 NIR and fluorescent dual mode imaging of clone 225 conjugated nanorose targeted to A431 cancer cells

5.4 Discussion

5.4.1 Packing of optical and magnetic functionality, along with antibody linker and antibody in a small overall particle size

5.4.2 Tuning antibody conjugation on nanoroses and the hydrodynamic sizes

5.4.3 Highly selective targeting observed by NIR and fluorescence duel model imaging

5.4.4 Effects of particle curvature and shape on delivery, imaging and therapy

xi

5.5 Conclustions

5.6 Supporting information

5.6.1 Calculating antibody numbers on spherical particle: circular assumption

5.6.2 Calculating antibody numbers on spherical particle: rectangular assumption

5.7 References

Chapter 6 Conclusions

6.1 Small multifunctional nanoclusters (nanoroses) for targeted cellular imaging and therapy

6.2 Growth of textured thin Au shells on iron oxide nanoparticles............186

6.3 Tuning the nucleation and growth of Au shells on iron oxide nanoclusters

6.4 Selective targeting of antibody conjugated multifunctional nanoclusters (nanoroses) to epidermal growth factor receptors in cancer cells......188

6.5 References

Bibliography

–  –  –

Table 2.S1: Multiple batches of nanorose to illustrate reproducibility in the particle size distribution by dynamic light scattering

Table 3.1: Elemental analysis of gold coated iron oxide nanoparticles made from a total Au/Fe mass ratio of 0.

50 with different iterations

Table 3.2: Mean size and standard deviation of textured thin gold coated iron oxide nanoparticles

Table 3.3: Elemental analysis of textured thin gold coated iron oxide nanoparticles by AAS

Table 3.4: Mean size and standard deviation of textured thin gold coated iron oxide nanoparticles

The effects of Au3+ precursor addition profiles

Table 3.5:

Table 3.6: Particle properties for different classes

Table 3.S1: Mean size and standard deviation of gold coated iron oxide nanoparticles in Figure 3.

2

Table 3.S2: Mean size and standard deviation of textured thin gold coated iron oxide nanoparticles

Table 3.S3: Calculated centrifugation speed for sedimentation of gold coated iron oxide nanoparticles

Table 3.S4: Detailed elemental analysis results of Fe and Au before and after centrifugation

Table 3.S5: Mass balance sheet of Fe and Au after centrifugation

Table 4.1: Properties of the particles synthesized at different initial pH values stabilized with 400 µg/ml mPEG-SH

–  –  –

initial pH values

Table 4.3: Particle properties for different classes of particles all with an added gold to iron mass ratio of 0.

5

Table 4.S1: This table contains data for the size distributions for the particles

–  –  –

the same data as shown in graph form in Figure 4.4f

Table 4.S2: This table contains data for the size distributions for the particles produced by using a starting pH of 8.

6 and a Au3+/Fe ratio of 0.5. This is the same data as shown in graph form in Figure 4.5a

Table 4.S3: This table contains data for the size distributions for the particles

–  –  –

the same data as shown in graph form in Figure 4.5c

Table 4.S4: Summary of the reaction conditions and the properties of the resulting particles for a Au3+/Fe feed ratio of 0.

5

–  –  –

diameter by dynamic light scattering

Table 5.2: Measured Extinction Coefficient at 755 nm of Thick Gold Coated Iron Oxide Nanoparticles and Nanorose particles

Table 5.3: Occupied surface area per Ab on nanorose and Au sphere.

...........169 Table 5.S1: Component of Abs conjugated nanorose particles

–  –  –



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