The Synthesis and Behavior of Positive and Negatively Charged Quantum Dots THESIS Presented in Partial Fulfillment of the Requir

The Synthesis and Behavior of Positive and Negatively Charged Quantum Dots

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Andrew Paul Zane

Graduate Program in Chemistry

The Ohio State University

2011

Master's Examination Committee:

Professor Prabir K. Dutta, Advisor

Professor Susan V. Olesik

Andrew Paul Zane

2011

Abstract

The synthesis, characteristics, and macrophage uptake of CdSe/ZnS core/shell quantum dots were studied. Insights into the mechanism of nucleation and growth of the quantum dots were gained by performing in-situ fluorescence spectroscopy during a microwave synthesis. The size and surface charge of quantum dots capped by 3- mercaptopropionic acid (3-MPA, negatively charged) and thiocholine (positively charged) were characterized by dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Finally, macrophage uptake studies were performed by Amber Nagy via flow cytometry to determine the level of quantum dot association with murine alveolar macrophages, and to determine a possible uptake pathway into the cells.

The mechanism for the CdSe/ZnS synthesis was determined by an in-situ fluorescence experiment. A fast nucleation step occurred, resulting in small CdSe seed nanoparticles which were protected from aggregation by the 3-MPA. Upon microwave heating, these caps were removed from the surface and began to deteriorate. The CdSe cores underwent Ostwald ripening in which smaller particles dissolved and provided free ions to increase the size of the larger particles. After this period, free zinc in the solution reacted with sulfur, freed from MPA decomposition, to form a ZnS shell around the CdSe core. The ZnS shell passivated the surface of the CdSe core, resulting in increased quantum yield. A maximum quantum yield of 22% was attained after 80 minutes of microwave heating, after which it began to decrease. ii

Both MPA and thiocholine coated quantum dots were stable in water and in

Roswell Park Memorial Institute (RPMI) media. MPA coated quantum dots aggregated in fetal bovine serum and serum free media, but remained stable. In both cases the surface charge was reduced, indicating the association of a species in the media with the quantum dots. We propose this was due to bovine serum albumin. Thiocholine coated quantum dots aggregated significantly in dilute fetal bovine serum and serum free media, and were not stable. In pure fetal bovine serum, the thiocholine quantum dots were stable, but had a negative surface charge. We propose that this sample was stable because the concentration of bovine serum albumin was high enough to fully coat and protect the quantum dots.

MPA coated quantum dots quickly associated with macrophages, and followed a scavenger receptor uptake pathway. This association was found to be charge dependent; less negatively charged quantum dots did not associate as strongly with the macrophages.

Positive and aggregated quantum dots did not associate as significantly, though some uptake was observed. This uptake was not via the scavenger receptor pathway.

iii

Dedicated to my parents Betty and Levi Zane

Acknowledgments

I would first like to acknowledge my advisor Dr. Prabir Dutta, who has tried his best to whip me into shape and is responsible for overseeing what I feel has been a time of tremendous personal growth. Each year I can look back and see how far I have gone; this distance would be much shorter without the guidance of Dr. Dutta. I look forward to continuing to work with and learn from him over the next few years.

I want to thank my lab-mates and co-workers, especially my friends Dedun

Adeyemo, Kevin Cassidy, Betsy Heck, Michael Severance, Suvra Mondal, Prasenjit Kar, and Govindhan Maduraiveeran who have all made my time in the lab and in Columbus better. Also: Jeremy White, Supriya Sabbani, Xiaogan Li, Weizhen Xiong, Brian Peebles,

Joselyn Del Pilar, Max Mullen, and Chenhu Sun. I want to thank William Schumacher, who developed the synthesis I use in this thesis and provided helpful guidance in getting started. I would also like to acknowledge Dr. James Waldman and Amber Nagy who are responsible for the cell uptake studies.

Last but not least I am grateful for my friends and family, who give me plenty of distractions from chemistry. I especially want to thank my loving and supportive girlfriend Nicolette Hicks for keeping me happy and sane over the last year. I look forward to going home every day to her and our cat Princess.

Vita

1985…………………………………………Born: Cincinnati, Ohio

2003...... Graduated Wilmington High School

Wilmington, Ohio

2008...... B.S. Chemistry, Wright State University

Dayton, Ohio

2008 to present ...... Graduate Teaching Associate, Department

of Chemistry, The Ohio State University

Columbus, Ohio

Fields of Study

Major Field: Chemistry

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vi

List of Tables ...... x

List of Figures ...... xi

Chapter 1: Introduction ...... 1

1.1 Quantum Dots ...... 1

1.2 Synthesis of Quantum Dots ...... 3

1.3 Particle Stability ...... 4

1.4 Light Scattering Measurements ...... 7

1.4.1 Dynamic Light Scattering (DLS) ...... 8

1.4.2 Electrophoretic Light Scattering (ELS) ...... 13

1.5 Quantum Dot Uptake ...... 20

1.6 Research Focus ...... 21

Chapter 2: Experimental Description...... 25

2.1 Synthesizing Negatively Charged Quantum Dots ...... 25 vii

2.1.1 Materials ...... 25

2.1.2 Precursor Solutions ...... 25

2.1.3 Reaction ...... 27

2.2 Synthesizing Positively Charged Quantum Dots ...... 28

2.2.1 Materials ...... 28

2.2.2 Thiocholine Solution ...... 28

2.2.3 Ligand Exchange ...... 28

2.3 Characterization ...... 29

2.3.1 Optical Characterization ...... 29

2.3.2 Size and Charge ...... 30

2.3.3 pH Titration ...... 30

2.3.4 Behavior in Biological Media ...... 31

2.4 In-Situ Fluorescence Study of Reaction Mechanism ...... 31

2.5 Quantum Dot Association with Macrophages ...... 33

Chapter 3: Results ...... 35

3.2 Optical Properties ...... 40

3.3 pH Titration ...... 41

3.4 Size and Charge in Biological Media ...... 43

3.4.1 Media Only ...... 44

viii

3.4.2 3-MPA Coated Quantum Dots ...... 47

3.4.3 Thiocholine Coated Quantum Dots ...... 51

3.5 Surface Charge in Biological Media ...... 54

3.5.1 Media Only ...... 55

3.5.2 MPA Coated Quantum Dots ...... 61

3.5.3 Thiocholine Coated Quantum Dots ...... 69

3.6 Quantum Dot Association with Macrophages ...... 76

Chapter 4: Discussion and Conclusions ...... 80

4.1 Mechanism of CdSe/ZnS Microwave Synthesis ...... 80

4.2 Optical Properties ...... 81

4.3 Physical Properties of Quantum Dots in Water ...... 82

4.4 Size of Quantum Dots in Biological Media ...... 83

4.5 Surface Charge of Quantum Dots in Biological Media ...... 84

4.6 Quantum Dot Association with Macrophages ...... 86

4.7 Comparison to Literature ...... 87

4.8 Conclusions ...... 89

References ...... 91

List of Tables

Table 1: List of RPMI 1640 Media Ingredients56 ...... 23

Table 2: Comparison of Abundant Proteins in FBS57 ...... 23

Table 3: Summary of DLS Volume Distribution Size Results ...... 43

Table 4: Summary of Zeta Potential Data...... 54

List of Figures

Figure 1: DLVO Theory: Stable Conditions ...... 6

Figure 2: DLVO Theory: Aggregation ...... 7

Figure 3: Diagram of a Malvern DLS Instrument ...... 9

Figure 4: Typical Correlogram ...... 10

Figure 5: Typical Intensity Distribution ...... 12

Figure 6: Typical Volume Distribution...... 13

Figure 7: Diagram of a Malvern ELS Instrument ...... 14

Figure 8: Hypothetical Reference (Top) and Scattered (Bottom) Waves ...... 15

Figure 9: Phase Shift of Scattered Wave from Reference Wave Over Time ...... 16

Figure 10: Typical ELS Phase Plot ...... 19

Figure 11: Typical Zeta Potential Distribution Plot ...... 19

Figure 12: Fluorolog 3 Connected to Discover SP Microwave ...... 32

Figure 13: Fiber Optic Cable Inserted Into Camera Port ...... 32

Figure 14: Time dependence of fluorescence spectra during QD synthesis ...... 36

Figure 15: QD synthesis stopped at various times, cooled, and diluted ...... 36

Figure 16: Time dependence of fluorescence spectra during QD synthesis ...... 37

Figure 17: Time Dependence of Fluorescence Spectra During QD Synthesis Without Zn

...... 39

Figure 18: UV/Visible Spectra of MPA and thiocholine coated quantum dots ...... 40 xi

Figure 19: Photoluminescence spectra of MPA and thiocholine coated quantum dots .... 41

Figure 20: pH Titration of MPA Coated Quantum Dots ...... 42

Figure 21: pH Titration of Thiocholine Coated Quantum Dots ...... 42

Figure 22: RPMI Media - Volume Distribution ...... 44

Figure 23: FBS Media - Volume Distribution ...... 45

Figure 24: FBS 10% Media - Volume Distribution ...... 45

Figure 25: Serum Free Media - Volume Distribution ...... 46

Figure 26: MPA Quantum Dots in Water - Volume Distribution ...... 47

Figure 27: MPA Quantum Dots in RPMI - Volume Distribution ...... 48

Figure 28: MPA Quantum Dots in FBS - Volume Distribution ...... 49

Figure 29: MPA Quantum Dots in FBS 10% Media - Volume Distribution...... 49

Figure 30: MPA Quantum Dots in Serum Free Media - Volume Distribution ...... 50

Figure 31: Thiocholine Quantum Dots in Water - Volume Distribution ...... 51

Figure 32: Thiocholine Quantum Dots in RPMI - Volume Distribution ...... 52

Figure 33: Thiocholine Quantum Dots in FBS - Volume Distribution ...... 52

Figure 34: Thiocholine Quantum Dots in FBS 10% Media - Volume Distribution ...... 53

Figure 35: Thiocholine Quantum Dots in Serum Free Media - Volume Distribution ...... 54

Figure 36: RPMI Media Phase Plot ...... 55

Figure 37: RPMI Media Zeta Potential Distribution ...... 56

Figure 38: FBS Media - Phase Plot ...... 57

Figure 39: FBS Media - Zeta Potential Distribution ...... 57

Figure 40: FBS 10% Media - Phase Plot ...... 58

xii

Figure 41: FBS 10% Media - Zeta Potential Distribution ...... 59

Figure 42: Serum Free Media - Phase Plot ...... 60

Figure 43: Serum Free Media - Zeta Potential Distribution ...... 60

Figure 44: MPA Quantum Dots in Water - Phase Plot ...... 61

Figure 45: MPA Quantum Dots in Water - Zeta Potential Distribution ...... 62

Figure 46: MPA Quantum Dots in RPMI - Phase Plot ...... 63

Figure 47: MPA Quantum Dots in RPMI - Zeta Potential Distribution ...... 63

Figure 48: MPA Quantum Dots in FBS - Phase Plot...... 64

Figure 49: MPA Quantum Dots in FBS - Zeta Potential Distribution ...... 65

Figure 50: MPA Quantum Dots in FBS 10% Media - Phase Plot ...... 66

Figure 51: MPA Quantum Dots in FBS 10% Media - Zeta Potential Distribution ...... 66

Figure 52: MPA Quantum Dots in Serum Free Media - Phase Plot ...... 67

Figure 53: MPA Quantum Dots in Serum Free Media - Zeta Potential Distribution ...... 68

Figure 54: Thiocholine QDs in Water - Phase Plot ...... 69

Figure 55: Thiocholine QDs in Water - Zeta Potential Distribution ...... 70

Figure 56: Thiocholine Quantum Dots in RPMI - Phase Plot ...... 71

Figure 57: Thiocholine Quantum Dots in RPMI - Zeta Potential Distribution ...... 71

Figure 58: Thiocholine Quantum Dots in FBS - Phase Plot ...... 72

Figure 59: Thiocholine Quantum Dots in FBS - Zeta Potential Distribution ...... 73

Figure 60: Thiocholine Quantum Dots in FBS 10% Media - Phase Plot ...... 74

Figure 61: Thiocholine Quantum Dots in FBS 10% Media - Zeta Potential Distribution 74

Figure 62: Thiocholine Quantum Dots in Serum Free Media - Phase Plot ...... 75

xiii

Figure 63: Thiocholine Quantum Dots in Serum Free Media - Zeta Potential Distribution

...... 76

Figure 64: Comparison of cell internalization of thiocholine and MPA coated quantum

dots in various media ...... 78

Figure 65: 3-Mercaptopropionic Acid ...... 82

Figure 66: Thiocholine ...... 83

xiv

Chapter 1: Introduction

1.1 Quantum Dots

Quantum dots are nano-scale semiconductors that fluoresce when excited with

UV light, due to the quantum confinement effect. In semi-conducting materials, an excited-state consists of an electron excited to the conduction band, and a hole left behind in the valence band. This is also known as an exciton. The electron and the valence-level hole it leaves behind have a natural spatial separation, called the Bohr exciton radius. If a particle is made smaller than the Bohr exciton radius, the confinement of the exciton will lead to an increase in the energy required to excite an electron into the conduction band, the band gap energy. As the exciton is constrained further, the band gap energy continues to increase. This phenomenon was initially described in 1982 by Brus1. The Bohr exciton radius is typically on the order of 10 nm, so producing particles of this size or smaller will result in fluorescence at various wavelengths. Using this strategy with various semi- conducting materials, quantum dots have been made which fluoresce across the entire visible spectrum2–5 and in the infrared6,7.

To control the size, and thus the optical properties, of quantum dots the nucleation and growth must be controlled. Nucleation and growth of nanoparticles is typically viewed as an initial fast growth that is reaction-rate limited, followed by slower diffusion- limited growth8. The exact mechanisms of nucleation and growth have proven to be difficult to assess, and vary depending on characteristics of the reaction solution. The 1 growth rate and ultimate size of the particles can be controlled by using varying amounts of stabilizing ligands9.

In addition to core quantum dot material, shells are typically grown around them which have been found to enhance the fluorescence of the core particle10. The quantum yield, the percentage of photons emitted for each photon absorbed, is affected by the quality of the quantum dot surface. Defects in the surface create trap states that result in non-radiative decay and a broad red-shifted trap-state emission peak11. Each of these reduces the quantum yield of the quantum dot.

In aqueous and organic solutions, quantum dots are stabilized by adding an appropriate organic capping agent, which can stabilize the particle sterically or electrostatically. The charges and functional groups on these caps dictate how quantum dots interact with their environment12,13. Other constituents in their environment may bind to quantum dots, for example humic substances in soil or water14. This could drastically change the surface characteristics, and thus the behavior, of the quantum dots.

Because of this, careful attention must be paid to what the quantum dots will encounter and how they will be affected.

Quantum dots are of interest to many fields. In biology and medicine, they have the potential to replace standard organic dyes. Quantum dots show less photo-bleaching and degradation, have more discreet emission wavelengths, and their wide absorption profile enables them to be coupled with multiple dyes or other quantum dots with different emission wavelengths6,15,16. In the development of medications targeted for specific areas of the body or specific types of cells, quantum dots can be useful through

2 bio-conjugation to track the destination of the molecule17,18. Long-lived quantum dots can be used to track the long term development and spread of cancers better than organic dyes6,19–21. Materials scientists are using quantum dots to sensitize solar cells, develop low cost imaging sensors, and make improved LED lighting that can be used in screens, monitors, and light bulbs5,22–27.

In spite of their promising characteristics, applications are limited due to high cost. A review of the quantum dot market published in 200928 listed prices ranging from

$3,000 to $10,000 per gram. Simple, highly scalable methods of production must be developed to lower prices and make commercial quantum dot utilization more reasonable.

1.2 Synthesis of Quantum Dots

Organic based methods of quantum dot production are well developed and yield high quality quantum dots with quantum yields between 50-80 %10. These typically involve the rapid injection of dimethyl cadmium or cadmium oxide with other organometallic reagents into hot coordinating solvents such as trioctylphosphine (TOP) and trioctylphosphine oxide (TOPO). Despite their high quality, organically prepared quantum dots are water insoluble and not appropriate for use in biological research.

Aqueous based methods of quantum dot synthesis yield water soluble quantum dots that are suitable for use in biological research. In these methods, cadmium chalcogens (Se, Te) are nucleated by reacting cadmium salts (CdCl2) with NaH(Se, Te) in the presence of a thiol, which acts as a passivating ligand9,29. These reaction methods

3 have the benefit of being safer, less expensive, and yielding water soluble particles.

However, the quantum yield of aqueous prepared quantum dots are typically lower than organic methods.

Microwave irradiation has been found to decrease the reaction time of quantum dot synthesis and increase the quantum yield of the product. These methods are simple and have the potential for easy scalability, as high quality microwave reactors minimize thermal gradient effects30. Microwave synthesis methods have produced CdTe quantum dots with quantum yields up to 60% after just 5 minutes of irradiation4. Further advancements have enabled the microwave production of more complex core/shell materials including CdSe(S)31, ZnSe(S)32, and CdSe/ZnS33.

1.3 Particle Stability

One issue with nanoparticle production, and thus quantum dot production, is the tendency of the particles to aggregate. This is undesired, as aggregated nanoparticles no longer have the intended behavior. Nanoparticle solutions can be stabilized by steric or electrostatic forces. Essentially, the particle must be coated with a capping agent that is either large enough to physically block the particles from coming together, or is charged enough that the particles will electrostatically repulse each other. This work will focus on electrostatic repulsion.

Electrostatic repulsion is a result of particles having the same charge on their surface. The overall charge on a particle’s surface is called the zeta potential. The larger the magnitude of the zeta potential, the more stable the particles will be. This is explained by DLVO theory, named for Derjaguin, Laundau, Verwey, and Overbeek34,35.

DLVO states that the net potential energy function of a system of particles, VT, is given by

VT=VA+VR+VS where VA is Van der Waals attraction, the natural tendency for the particles to aggregate, and VR is the electrostatic repulsion between particles. VS accounts for solvent effects, but these are typically small enough to be ignored. VA and VR are modeled as follows:

where A is the Hamaker constant, D is particle separation, ε is electric permittivity of the solvent, a is particle radius, ζ is the zeta potential, and k is the Debye screening length, based on the ionic strength of the medium.

The relative contributions of VA and VR are demonstrated in Figure 1 and Figure

2. In Figure 1, the repulsive force is high and overpowers the Van der Waals attractive forces. The net energy is positive, and increases as the particles come closer together.

These particles are driven away from each other. In Figure 2, the repulsive force has been lowered, and there is no barrier to the particles coming together. There is a small secondary minimum in energy, allowing loosely bonded aggregates to form.

The equation for VR illustrates that smaller particles are less stable, as the repulsive force is directly proportional to the particle radius. Charge is even more significant, as the repulsive force is proportional to the square of the zeta potential.

Another major factor is the ionic strength of the medium, which affects the Debye screening length. Any of these could lead to an unstable system like that shown in Figure

2 rather than the stable system in Figure 1.

Figure 1: DLVO Theory: Stable Conditions

Figure 2: DLVO Theory: Aggregation

1.4 Light Scattering Measurements

Because of their optical properties, characterization of quantum dots typically includes UV-visible absorption and fluorescence spectroscopy. These measurements indicate the optical properties and overall quality of the quantum dots, but they do not characterize the size behavior of the quantum dots in a solution. To obtain this information, dynamic light scattering (DLS) and electrophoretic light scattering (ELS) are used.

1.4.1 Dynamic Light Scattering (DLS)

DLS is a light scattering technique used to determine the size of a population of particles. The size is determined by measuring the Brownian motion of the particles, which is related to the size using the Stokes-Einstein equation:

where D is the diffusion coefficient (measure of Brownian motion), kb is Boltzmann’s constant, T is the temperature in Kelvin, η is the viscosity of the solution, and d is the hydrodynamic radius. DLS measures the hydrodynamic radius rather than the true radius.

Various factors such as ionic strength, particle shape, and surface characteristics can affect how the particle diffuses through the medium, resulting in a calculated diameter that is typically larger than the true value.

To measure the Brownian motion of a particle, represented by the diffusion coefficient, the DLS instrument measures the intensity of light scattered by a moving particle. The basic premise is that the intensity of the scattered light will change relative to how fast the particle is traveling.

Figure 3 shows a diagram for a Malvern Zetasizer. The instrument directs light from a laser into the sample cell, and measures the intensity of the light scattered by particles at a detector set to a specific angle from the laser. The intensity of scattered light hitting this point is measured at rapid intervals, on the order of nano to microseconds. A correlator processes the signal which is then interpreted by computer software.

Figure 3: Diagram of a Malvern DLS Instrument

Using the intensity of light at each time interval, the instrument constructs a correlation function:

〈 ( ) ( )〉 ( ) 〈 ( )〉 where I(t) is the starting intensity and I(t+τ) is the intensity after an interval of time, τ.

Because the correlation function is normalized, it should always be between 0 and 1, with

1 being complete correlation, and 0 being no correlation.

Figure 4 shows a typical correlogram obtained with DLS. The correlation begins with a flat line at a coefficient of 0.9. This means that within the early time frame, none of the particles have moved enough to significantly change the intensity of light reaching the detector. Soon after this, as the particles move, the correlation sharply drops to 0. This loss of correlation will happen earlier in a correlogram with smaller, faster moving particles, and later in a correlogram with larger, slower moving particles.

Figure 4: Typical Correlogram

In a simple system with one population of particles, the correlation function can be fit with a single exponential decay:

( ) ( ( )) where A is the baseline of the correlation function, and B is the intercept of the correlation function. More complicated samples are fit with a sum of exponentials.

From the exponential fit, Γ is obtained which is then used to determine the diffusion coefficient by the equations

( )

where D is the diffusion coefficient, n is the refractive index of the particle material, λ is the wavelength of the scattering light, and θ is the scattering angle. The calculated diffusion coefficient can then be used with the Stokes-Einstein equation shown previously to determine the hydrodynamic diameter.

The distribution obtained from the direct conversion from correlation function to size is called an intensity distribution. Figure 5 shows a typical intensity distribution derived from the correlogram shown in Figure 4.

Figure 5: Typical Intensity Distribution

Intensity distributions are sometimes deceptive, because the intensity of scattering has a 106 dependence on the radius of the particle: larger particles will overshadow smaller particles. A small number of aggregated particles or dust particles may appear to be the most significant population in an intensity distribution, while the true make-up of the sample is a much larger number of smaller particles. This effect is somewhat present in Figure 5. There is a long tail to the right of the main distribution extending to 500 nm, along with a small population of particles around 4000 nm.

Mie theory36 provides a model for the relative scattering intensities of particles based on their size. To deal with skewed data, Mie theory has been used to convert from intensity distributions, where scattering intensity is proportional to r6, to a number distribution, where the signal is directly proportional to the radius.

The number distribution can also be deceptive, as noise early in the data can be over-interpreted, giving false distributions of very small particles. Number distributions can be converted to volume distributions, where the signal is proportional to r3. Being in

12 the middle, volume distributions are less prone to the problems of intensity and number distributions. Figure 6 shows a typical volume distribution converted from the intensity distribution above. The small peak at 5000 nm is not visible, and the long tail is mostly gone.

Figure 6: Typical Volume Distribution

1.4.2 Electrophoretic Light Scattering (ELS)

ELS is a light scattering technique used to measure the surface charge, or zeta potential, of a population of particles. ELS measures the electrophoretic mobility of a particle: the velocity a particle travels divided by the electric field being applied which is causing it to move.

The electrophoretic mobility can be determined through Phase Analysis Light

Scattering (PALS)37. Figure 7 illustrates the ELS set-up of a Malvern Zetasizer. The instrument passes light through the sample and compares it to a split reference beam. The light passing through the sample is scattered by particles in the sample and Doppler

13 shifted to a new frequency. The frequency shift is dependent on the speed of the scattering particle:

( )

ωs is the frequency shift, q is the momentum transfer vector, ve is the electrophoretic component of the velocity, and vc is velocity due to other causes, such as thermal convection. The momentum transfer vector is a constant value determined by the material’s refractive index, the wavelength of the laser light, and the scattering angle.

Figure 7: Diagram of a Malvern ELS Instrument

In PALS, the time derivative of the phase is measured. The scattered light is at a different frequency, so its phase will become more shifted from the reference beam over time. The rate of the phase shift is related to the frequency shift: the further the frequency is shifted, the faster the phase will shift. Figure 8 and Figure 9 demonstrate a hypothetical

PALS experiment. The top wave in Figure 8 is the original reference wave, with a frequency of 300 Hz. Below is a scattered wave, which has been Doppler shifted to 320

Hz.

Figure 8: Hypothetical Reference (Top) and Scattered (Bottom) Waves

Figure 9 illustrates what the phase shift plot would look like for this 20 Hz frequency shift.

Figure 9: Phase Shift of Scattered Wave from Reference Wave Over Time

By taking the derivative of the phase plot at any time, the frequency shift can be extracted. For example, at 0.005 seconds:

( )

The initial result is in radians. Dividing by 2π gives the original frequency shift of 20 Hz.

It is now apparent that the frequency shift, , is equivalent to the time derivative of the phase plot. An advantage of PALS becomes evident here: rather than having one frequency shift value to calculate one zeta potential, a large number of derivatives can be used to calculate a large number of zeta potentials to be averaged together. These measurements can also be taken very quickly, as the derivative can be calculated at any

16 point on the phase shift plot. The frequency shift in the earlier equation can now be replaced with the phase shift derivative.

( ) ( )

To continue towards the objective of determining a zeta potential, the electrophoretic velocity, ve, is related to the electrophoretic mobility, , and the applied electric potential, E.

Substituting for the electrophoretic velocity now gives an equation with which the measured phase shift derivative can be used to calculate the electrophoretic mobility:

( ) (〈 〉 ( ) )

Finally, the electrophoretic mobility is used with the Henry equation to determine the zeta potential:

( )

where ε is the dielectric constant, ζ is the zeta potential, η is the viscosity of the medium, and f(ka) is Henry’s function.

In Henry’s function, k is the Debye screening length and a is the particle radius.

In most cases, the Smoluchowski model is used, which approximates f(ka) to be 1.5. The

Smoluchowski model is relevant for samples where the Debye screening is small compared to the particle radius (high ionic strength, high dielectric constant media).

When the media has a low ionic strength, and the Debye screening is large compared to the particle radius, the Huckel model is used, which approximates f(ka) to be 1. 17

Typically, aqueous solutions will use Smoluchowski while non-polar solutions will use the Huckel model.

The raw data obtained by the Zetasizer Nano is displayed as a phase plot similar to Figure 9, showing the phase shift versus time. The Zetasizer Nano rapidly reverses the electric field every 0.05 seconds, yielding a saw tooth pattern. An example phase plot from the Zetasizer is shown in Figure 10. The first half of the plot is the fast field reversal. Electrophoretic mobility, used here to determine the zeta potential, reaches terminal velocity on a microsecond time scale, so these measurements can be taken quickly. The fast field measurements are not long enough to Fourier transform a zeta potential distribution, so the slow field reversal in the second half of the plot is used to determine the distribution.

While the slow field reversal allows for a distribution to be transformed, the mean zeta potential that it is centered on will be shifted due to electro-osmosis effects. Electro- osmosis effects develop on the millisecond timescale, so they primarily affect the slow field reversal and not the fast field reversal. The more accurate mean zeta potential from the fast field reversal is used to shift the distribution to the correct value, which is then displayed in a distribution plot like the one seen in Figure 11.

Figure 10: Typical ELS Phase Plot

Figure 11: Typical Zeta Potential Distribution Plot

1.5 Quantum Dot Uptake

As discussed previously, quantum dots are of interest in biology for tracking and imaging. While they have proven to be useful, the exact mechanisms of cell uptake are only recently being characterized. A recent study38 found that negatively charged

CdSe/ZnS quantum dots were efficiently taken into cells while neutral and positively charged quantum dots were not. Because of low uptake in the neutral and positive quantum dots, only negative quantum dots were studied further. Negatively charged quantum dot uptake was found to be regulated primarily by the LDLR/SR (low density lipoprotein receptor/scavenger receptor) pathway. Scavenger receptors are known to recognize acetylated LDL, which has a negative charge. The size of human LDL is 24-26 nm39, similar to the hydrodynamic radius of quantum dots (27 nm in the cited study).

Because of their similar size and charge, it seems reasonable that these receptors would recognize negatively charged quantum dots. This suggests that quantum dot uptake is dependent on size and charge. Other studies40,41 have found that positively charged nanoparticles follow a clathrin dependent uptake path, but these were larger and did not include quantum dots.

1.6 Research Focus

In this thesis, a previously reported33 microwave synthesis of CdSe/ZnS quantum dots is further studied to determine the reaction mechanism and improve the quantum yield of the resulting particles. Optical characterization is a powerful tool for determining the quality of quantum dots. A decreased quantum yield indicates that the surface is not well protected and contains many trap states, attributed to surface defects. Optical characterization can also indicate particle size, as the emission wavelength is dependent on the size of the core quantum dot. Shifting emission wavelengths indicate growing or shrinking particles.

Typically, these optical measurements are made after a synthesis is complete. The solution must be cooled, removed from the reaction vessel, and placed in a cuvette for measurement in a fluorimeter. In this research, a fiber-optic probe is used to couple a fluorimeter to a microwave oven, so that these optical properties may be observed in real- time during the reaction. This offers insights into the formation mechanisms. Having this information will lead to improvements in the synthesis.

Once they have been synthesized, the behavior of quantum dots in various conditions is an important consideration, particularly for biological research. Quantum dots have been used in drug delivery42–44, tracking45–48, and bio-imaging49–51. If the particles change from their intended behavior, these applications will be affected.

Nanoparticle behavior is dominated by surface characteristics. If a prepared nanoparticle is modified by molecules it encounters in solution, its characteristics and behavior may change from what was intended. In the complex mixtures encountered in biological

21 systems, there can be thousands of different species present. It seems plausible that some of these may interact with nanoparticles. Albumin in particular has been shown to interact with a wide range of nanoparticles52–55.

Thiocholine and MPA coated quantum dots were examined in several media: water, Roswell Park Memorial Institute Media 1640, fetal bovine serum, and X-Vivo 15 serum free media. These media are all used in biological research and applications. The effects of these media on quantum dots were observed by measuring their size and surface charge, both of which affect nanoparticle behavior.

Roswell Park Memorial Institute media (RPMI) is a well-defined mixture of amino acids, salts, and vitamins56, summarized in Table 1. Fetal bovine serum (FBS) is less well defined because it is extracted from the blood of a fetal calf. Variability from animal to animal means that concentrations will not always be the same. The large number of components in FBS makes characterization difficult. By using liquid chromatography and mass spectrometry, some proteins have been identified and quantified57. The largest component in the analysis was serum albumin. A summary of the proteins found and their relative amounts is found in Table 2. Batch-to-batch variations in concentrations, lack of characterization, and animal welfare concerns have led to a demand for synthetic alternatives to FBS. X-Vivo 15 Serum Free Media is one such media intended to replace FBS. The formulation of X-Vivo 15 is proprietary, but it is known to contain glutamine and human insulin, albumin, and transferrin. Because it is intended to replace FBS, it seems likely that the concentrations used are similar to those found in FBS.

RPMI 1640 Ingredients Inorganic Salts Calcium Nitrate, Magnesium Sulfate, Potassium Chloride, Sodium Bicarbonate, Sodium Chloride, Sodium Phosphate Dibasic Amino Acids Arginine, Asparagine, Aspartic Acid, Cystine, Glutamic Acid, Glutamine, Glycine, Histidine, Hydroxy-L-Proline, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine Vitamins Biotin, Choline Chloride, Folic Acid, Inositol, Niacinamide, Aminobenzoic Acid, Pantothenic Acid, Pyridoxine, Riboflavin, Thiamine, B12 Other Glucose, Glutathione, Phenol Red Table 1: List of RPMI 1640 Media Ingredients56

Table 2: Comparison of Abundant Proteins in FBS57

As previously stated, the size and charge of quantum dots affect their uptake by cells. It is expected that the thiocholine and MPA coated quantum dots synthesized here will likely not have the same uptake efficiency or pathway. In addition to this, the various media that the quantum dots are exposed to may change their size and charge, and subsequently change their interactions with cells. The effects of these media on quantum dot-cell association were studied through flow cytometry experiments.

Thiocholine and MPA coated quantum dots were dispersed in RPMI, RPMI+FBS, and X-Vivo 15 serum free media. Murine alveolar macrophages were exposed to these mixtures, and flow cytometry was used to measure the amount of quantum dot association, indicating whether or not the macrophages have taken up the particles.

Additional experiments included addition of polyinosinic acid (Poly-I) to thiocholine and

MPA coated quantum dots in each media. Poly-I associates with the LDLR/SR pathway that negative quantum dots are known to enter other cell lines through. A hypothesis of the LDLR/SR pathway was examined for the MPA coated quantum dots in this cell line, and whether thiocholine coated quantum dots follow a different mechanism was explored.

In summary, the objective of this thesis is to determine the nucleation mechanism of CdSe/ZnS core/shell quantum dots in a microwave synthesis, the size and charge behavior of those quantum dots in different biological media, and how size and charge influence quantum dot uptake by macrophage cells.

Chapter 2: Experimental Description

2.1 Synthesizing Negatively Charged Quantum Dots

Cadmium selenide core/zinc sulfide shell quantum dots were produced with a negatively charged capping agent, 3-mercaptopropoionic acid (MPA), in a previously reported microwave synthesis33.

2.1.1 Materials

Cadmium chloride hemipentahydrate (CdCl2·2.5 H2O, >98 %) and sodium borohydride (NaBH4, 99 %) were obtained from Aldrich (Milwaukee, WI, USA). Zinc chloride (ZnCl2, 99.99 %), 3-mercaptopropionic acid (MPA), and selenium powder (Se,

99.5+ %, 200 mesh) were obtained from Acros (Geel, Belgium). Sodium hydroxide

(NaOH) and ammonium hydroxide (NH4OH, 28 – 30 %) were obtained from

Mallinckrodt Chemicals (Phillipsburg, NJ, USA). All chemicals were used without further purification. The H2O used in this study was purified by a Barnstead NANOpure

Infinity ultrapure water system (Dubuque, IA, USA).

2.1.2 Precursor Solutions

Cadmium/MPA

A 250 mL solution of 1.05 mM Cd and 5.26 mM MPA was prepared by adding

60 mg of cadmium chloride hemipentahydrate and 114.5 μL of MPA to approximately

225 mL water. The pH was adjusted to 9.5 with 1 M NaOH, and the volume was adjusted

25 to 250 mL using a volumetric flask. The solution turned from clear to white then back to clear while the NaOH was added. The final solution was stored in a plastic bottle and wrapped in aluminum foil to protect from light.

+ Zn(NH3)4

+ A 25 mL solution of 26.67 mM Zn(NH3)4 was prepared using anhydrous zinc chloride and ammonium hydroxide. The hygroscopic zinc chloride was dried in a vacuum oven for one hour at 200 °C and capped prior to weighing. To minimize error in weighing due to absorption of water from the air, approximately 90.9 mg of zinc chloride was quickly added to a previously weighed beaker containing 20 mL of water. Ammonium hydroxide was then added drop wise. A white precipitate formed and then disappeared as more ammonium hydroxide was added. Once the solution was fully clear, the volume of the solution was adjusted to 25 mL in a volumetric flask. Typically more than 90.9 mg of zinc chloride was added during the quick measurement, so the final volume was adjusted with a pipette to achieve the correct concentration. The final solution was stored in a plastic bottle, wrapped in aluminum foil to protect from light, and stored at 4 °C.

NaHSe

152 mg of sodium borohydride was added to a glass test tube and chilled to 0 °C in an ethylene glycol/water bath. In quick succession, 2 mL of chilled water was pipetted into the test tube, followed by 158 mg of selenium powder. The test tube was capped with a rubber stopper with two needles sticking through; one of the needles was attached to a nitrogen gas tank, and the other was left open to vent. The test tube was placed back into the cold bath.

The reaction showed intense bubbling from the evolution of hydrogen gas, which lasted for approximately 30 minutes. By this time, the black selenium was reduced into the solution and a white precipitate formed. The bubbling gradually decreased until stopping after approximately 3 hours.

4 NaBH4 + 2 Se + 7 H2O → 2 NaHSe + Na2B4O7 + 14 H2

After completion of the reaction, the vial was quickly moved to a glove bag filled with nitrogen. 0.5 mL of the supernatant was quickly pipetted into a 50 mL 3-neck round bottom flask containing 24.5 mL of nitrogen saturated water. This yielded a 25 mL solution of 20 mM NaHSe. The flask was kept capped and nitrogen was bubbled though the solution. Over time, the presence of a red tint indicated that the selenium had been oxidized, and the solution was no longer useful. The solution typically lasted 12 to 18 hours.

2.1.3 Reaction

19 mL of the Cd/MPA solution was added to a 25 mL Erlenmeyer flask and vigorously stirred on a stir plate. 0.25 mL of the NaHSe solution was quickly added, resulting in an immediate yellow color due to CdSe formation. The solution was left to

+ nucleate for 1 hour. After nucleation, 0.75 mL of the Zn(NH3)4 solution was added. The final 20 mL solution was 1 mM Cd, 1mM Zn, 0.25 mM Se, and 5 mM MPA.

The solution was placed in a Discover SP (CEM Corp.) microwave system and heated for 90 minutes at 150 °C, with maximum power set to 200 watts. After completion the solution was cooled to 40 °C in the microwave with a stream of room temperature air, and was then stored in a glass vial wrapped in aluminum foil at 4 °C until needed.

2.2 Synthesizing Positively Charged Quantum Dots

Positively charged quantum dots were prepared by a ligand exchange using the previously prepared MPA capped particles.

2.2.1 Materials

Acetylthiocholine chloride (>99%) was obtained from Aldrich (Milwaukee, WI,

USA). Sodium hydroxide (NaOH) was obtained from Mallinckrodt Chemicals

(Phillipsburg, NJ, USA). The H2O used in this study was purified by a Barnstead

NANOpure Infinity ultrapure water system (Dubuque, IA, USA).

2.2.2 Thiocholine Solution

A 25 mM thiocholine solution was prepared through a hydrolysis reaction of acetylthiocholine chloride with 1 M NaOH. Progress of the hydrolysis reaction was monitored with an Accumet AB15 pH Meter (Fisher Scientific). 500 mg of acetylthiocholine chloride was dissolved in 80 mL of water and stirred. NaOH was added drop-wise until the pH significantly increased (pH = 10 to 11). The pH decreased as hydroxide ions were consumed in the reaction. As the pH decreased, more NaOH was added. This process continued until the pH stabilized. The final pH was adjusted to 9 using 1 M NaOH or HCl, and the final volume was adjusted to 100 mL.

2.2.3 Ligand Exchange

20 mL of MPA capped quantum dots were centrifuged at 200,000x g using a

Sorvall MT-150 Ultracentrifuge with S-50A rotor (Thermo Fisher) for 60 minutes. After this step, quantum dots were seen as a localized cloud at the bottom of the centrifuge

28 tube. A majority of the clear supernatant was removed without disturbing the quantum dots. The solution was replaced with water, sonicated, and stirred.

This procedure was repeated multiple times. Each time the quantum dots appeared more pellet-like after centrifuging, and became more difficult to re-disperse. After 4 washing cycles, the quantum dots came out of the centrifuge as a single pellet that did not re-disperse. At this point all of the water was removed, and 20 mL of the thiocholine solution was added. The quantum dots were re-dispersed by alternately sonicating and stirring until the solution was clear and colored with no pellet remaining at the bottom of the centrifuge tube.

2.3 Characterization

2.3.1 Optical Characterization

UV/Visible and fluorescence spectra were recorded on thiocholine and MPA coated quantum dots diluted 10:1 with water. UV/Visible spectra were recorded with a

Shimadzu UV-2501PC spectrophotometer. Fluorescence measurements were recorded with a Horiba Jobin-Yvon Fluorolog 3, using 2 nm slit widths for the emission and excitation monochromators, 0.3 second integration time, and a 375 nm excitation wavelength. Quantum yields were estimated by comparison of the fluorescence intensity of the sample to the intensity of rhodamine 6G. Both samples were prepared to have an absorbance of 0.02 units at 480 nm excitation wavelength. Rhodamine 6G is known to have a quantum yield of 95% at 480 nm. By comparing the fluorescence intensities, a quantum yield for the sample was calculated.

2.3.2 Size and Charge

A Zetasizer Nano ZS (Malvern) was used to determine the size and zeta potential of the quantum dots. The Nano ZS uses a 633 nm laser as its light source.

The refractive index of the material was set to 2.351; the room temperature value for ZnS and 633 nm light (have reference). The absorption was left at the default value of 0.01.

For size measurements, a 173° backscatter angle was used for collecting scattered light. The instrument was set to automatically determine the number of runs, the run duration, and the optimal focal point for each sample. The analysis model was set to general purpose, with default size limits of 0.4-10000 nm diameter. The Smoluchowski model was used to fit the correlation functions to intensity distributions. Intensity distributions were converted to volume distributions by a software conversion based on

Mie theory. Three replicate measurements were taken for all samples and averaged. All particle sizes are given as diameters.

For zeta potential measurements, a forward angle of 12° was used for collecting light. The default Smoluchowski model was used. Each measurement was forced to contain 20 runs and to use the general purpose analysis. Three replicate measurements were taken for all samples and averaged.

2.3.3 pH Titration

The stability of both thiocholine and MPA coated quantum dots in water over a range of pHs was observed by using the Zetasizer Nano ZS with an attached MPT-2

Auto-titrator (Malvern). The settings and parameters used were the same as described previously. The starting concentration of quantum dots was 931 nM for size titrations and

186 nM for zeta potential titrations. The titrator used 1 M HCl to lower the pH by 0.3 units between each measurement. Three replicate measurements were taken for all samples and averaged.

2.3.4 Behavior in Biological Media

MPA and thiocholine coated quantum dots were diluted 1:1 with various media which are typical in biological experiments: fetal bovine serum (FBS); Roswell Park

Memorial Institute (RPMI) medium; a 90:10 mixture of RPMI/FBS; X-Vivo 15 Serum

Free Media; and water. Solutions were stirred at room temperature for 30 minutes after mixing.

DLS and ELS measurements were made to determine the size and zeta potential of the particles in each media, using the same configuration and settings described previously. Three measurements were taken for all samples and averaged.

2.4 In-Situ Fluorescence Study of Reaction Mechanism

In-situ fluorescence measurements were made with a Discover SP (CEM) microwave system during quantum dot synthesis. The Discover SP system is configured with a port hole for use with a camera attachment to image the sample during heating. By coupling this port hole to a fluorimeter, the fluorescence of the contents of the microwave chamber was measured during synthesis. Figure 12 shows the fiber optic attachment of the Fluorolog 3 fluorimeter running to the Discover SP microwave. Figure 13 shows a close-up of the fiber-optic inserted into the camera port inside of the microwave.

Fiber-Optic Exiting Sample Chamber of Fluorolog 3

Fiber-Optic Placed Through Camera Porthole

Figure 12: Fluorolog 3 Connected to Discover SP Microwave

Microwave Reaction Chamber

Fiber-Optic Cable Inserted In Camera Port

Figure 13: Fiber Optic Cable Inserted Into Camera Port 32

Batch runs were set up using the fluorimeter software to take approximately one spectrum every minute. A significant fluorescence near 500 nm was present when using the 375 nm excitation wavelength used in characterizing the synthesized quantum dots.

This peak was also observed when the microwave chamber was empty. Moving the excitation wavelength from 375 nm to 480 nm eliminated the background fluorescence of the chamber, but still excited quantum dot emission. A 6 nm band width was used for both the excitation and emission monochromators. The emission wavelength was scanned from 500 to 750 nm with an integration time of 0.2 seconds.

To determine the role of zinc and 3-MPA in the synthesis, subsequent trials were performed with each of these being subtracted from the initial reactant solution. The removal of 3-MPA resulted in immediate aggregation, so no microwave experiment was performed. Removal of zinc resulted in fluorescence intensity that was too low to measure at the reaction temperature. Several syntheses were performed, each time increasing the microwave irradiation time by 10 minutes. The reaction vial was then cooled to 10 °C before measuring the fluorescence spectrum.

2.5 Quantum Dot Association with Macrophages

The effects of size and charge on macrophage association with quantum dots were studied by Amber Nagy. Cell fluorescence (a measure of quantum dot association) was observed via flow cytometry. Quantum dots were dispersed in three biological media

(RPMI, 20% FBS in RPMI, and X-Vivo 15 serum free media). Murine alveolar macrophages were exposed to the quantum dots for 6 hours with and without Poly-I, then

33 harvested and analyzed for fluorescence in a FACScalibur flow cytometer with an excitation wavelength of 488 nm.

Chapter 3: Results

3.1 Mechanism of CdSe/ZnS Microwave Synthesis

Figure 14 shows a 3D plot of the time dependent fluorescence during microwave synthesis of CdSe/ZnS quantum dots. The x and y axes are emission wavelength and fluorescence intensity, and the z axis is time. Each spectrum represents approximately 1.5 minutes. The initial spectrum is prior to microwave heating. In the first 5 minutes, the intensity decreased as the reaction temperature increased. After this, two emissions began to be observed; a small peak at 502 nm, and the main quantum dot emission at 574 nm.

The 502 nm emission began to decrease after 20 minutes. The fluorescence of the main quantum dot emission reached a maximum intensity near 60 minutes and then began to decline. This seems to suggest that the quantum dots decreased in quality after 60 minutes, contrary to the literature suggestion of 90 minutes. In a subsequent experiment, the synthesis was stopped at 10 minute intervals, cooled, diluted 10:1 in water and measured in a quartz cuvette (Figure 15). The maximum fluorescence was at 80 minutes, closer to the ideal reaction time of 90 minutes suggested in the original development of this synthesis33 The decrease in intensity seen in the microwave experiment after 60 minutes was due to self-quenching58: the concentration of quantum dots after this time is too high to reliably measure their fluorescence.

60 Minutes, 574 nm

90 min

45 min

0 min 20 Minutes, 502 nm

Figure 14: Time dependence of fluorescence spectra during QD synthesis

1.0E+07 9.0E+06 10 Min 20 Min 8.0E+06 30 Min 7.0E+06 40 Min 50 Min 6.0E+06 60 Min 5.0E+06 70 Min 80 Min 4.0E+06 90 Min 100 Min 3.0E+06 110 Min Fluorescence Intensity Fluorescence 2.0E+06 1.0E+06 0.0E+00 400 500 600 700 800 Wavelength (nm)

Figure 15: QD synthesis stopped at various times, cooled, and diluted 36

Figure 16 shows a simplified view of the in-situ experiment, starting from the lowest fluorescence intensity 5 minutes into the experiment, and ending with the maximum fluorescence intensity 60 minutes into the experiment. Simultaneous growth of two emissions, one at 502 nm and one starting near 550 nm, occurs in the first 20 minutes of the synthesis. The 502 nm emission begins to decrease in intensity after 20 minutes.

The 550 nm emission increases throughout the synthesis and red-shifts to 574 nm.

60 Minutes, 574 nm

20 Minutes, 502 nm

Figure 16: Time dependence of fluorescence spectra during QD synthesis

We propose that the lower wavelength emission at 502 nm seen in Figure 14 was from a population of small CdSe particles. As the reaction progressed, these dissolved via

Ostwald ripening, depositing onto the larger CdSe particles emitting near 550 nm. These larger particles became the quantum dot cores. The emission increased in intensity and red-shifted as the reaction progressed. The initial red-shifting could be due to the core growing before the shell begins to form: as discussed in Chapter 1, as a quantum dot’s size increases, its band gap relaxes to a lower energy/higher wavelength. Later in the synthesis, the emission continues to red-shift due to the growth of the ZnS shell59.

The in-situ experiment was repeated in the absence of zinc ions, shown in Figure

17. Fluorescence was not intense enough to see at the reaction temperature. To increase intensity, the synthesis was interrupted at 10 minute intervals and cooled to 10 °C before measuring. Again, two emission peaks are visible near 500 nm and 550 nm. The 502 nm emission behaves similarly to the synthesis including zinc. It increases during the beginning of the synthesis and then disappears. The 545 nm emission increases in intensity for the first 30 minutes of the synthesis, and then also decreases. This is due to the quantum dots growing too large and becoming bulk CdSe. The solution had a dark red color, similar to bulk CdSe. This sample also shows a broad emission near 700 nm from trap-states.

10 Min 2.5E+04 20 Min 30 Min 40 Min 2.0E+04 50 Min 60 Min 70 Min 1.5E+04 80 Min 90 Min 100 Min 1.0E+04

Fluorescence Intensity Fluorescence 5.0E+03

0.0E+00 490 540 590 640 690 740 790 Wavelength (nm)

Figure 17: Time Dependence of Fluorescence Spectra During QD Synthesis Without Zn

The two experiments showed similar behavior for the first 30 minutes. Both had small peaks near 500 nm that quickly disappeared, and peaks starting near 550 nm that increased in intensity and red-shifted. After 30 minutes, the two experiments diverge. In the synthesis that included zinc, the intensity of the main quantum dot peak (550 nm) continued to increase. In the synthesis that did not include zinc, the intensity of this peak began to decrease. The synthesis without zinc also showed a more prominent trap state emission near 700 nm.

3.2 Optical Properties

Altering the capping ligand did not affect the optical properties of the quantum dots. MPA coated quantum dots had a maximum intensity of 5x107 at 558 nm.

Thiocholine coated quantum dots had a maximum intensity of 5x107 at 559 nm. These are well within the range of normal variations seen between different samples using this synthesis. The quantum yields were 22% for MPA coated quantum dots and 20% for thiocholine coated quantum dots.

0.16

0.14

0.12

0.1 Thiocholine 0.08 MPA

0.06 UV/Vis Absorbance UV/Vis

0.04

0.02

0 400 450 500 550 600 650 700 750 800 Wavelength (nm)

Figure 18: UV/Visible Spectra of MPA and thiocholine coated quantum dots

5.0E+07

Thiocholine 4.0E+07 MPA

3.0E+07

2.0E+07

1.0E+07 Photoluminescence (arbitrary units) Intensity Photoluminescence 0.0E+00 400 450 500 550 600 650 700 750 800 Wavelength (nm)

Figure 19: Photoluminescence spectra of MPA and thiocholine coated quantum dots

3.3 pH Titration

MPA coated quantum dots were stable in basic conditions. The titration, Figure

20, began with a pH of 10. The particles remained the same size until the pH of the solution was less than 6, after which they quickly began to agglomerate. Thiocholine coated quantum dots were stable in acidic conditions. The titration, Figure 21, began with a pH of 9. The particles aggregated, but remained stable through the final pH of 2.

Figure 20: pH Titration of MPA Coated Quantum Dots

Figure 21: pH Titration of Thiocholine Coated Quantum Dots

3.4 Size in Biological Media

As discussed in Chapter 1, the size and charge of nanoparticles is important to their behavior. Because biological media can be complicated mixtures (see Table 1 and

Table 2), it is possible that they will affect the properties of nanoparticles if a constituent associates with them. We report here the size of each media alone, thiocholine coated quantum dots in each media, and MPA coated quantum dots in each media. Table 3 summarizes all DLS size data. Sizes are based on volume distributions.

Media Alone Thiocholine QDs MPA QDs Media Size Count Rate Size Count Rate Size Count Rate (d, nm) (kcps) (d, nm) (kcps) (d, nm) (kcps) RPMI 1.0 35 20, 62* 1.7x103 8.2, 26* 4.6x103 RPMI/FBS 7.8 4.0x103 140, 1200* 1.0x105 54 2.3x103 FBS 6.8 4.0x104 7.0, 28* 8.6x104 7.8, 57* 4.9x104 X-Vivo 15 6.5 1.0 x103 3200** 5.7x104 12, 39* 1.5x104 Water - - 17 1.3x103 27 3.9x103 Table 3: Summary of DLS Volume Distribution Size Results * Multimodal distributions in the number distribution plot, including distributions extending past the range of the instrument likely from dust ** Wide distributions, indicating aggregation

3.4.1 Media Only

Roswell Park Memorial Institution Media

RPMI had the lowest size of the media observed, with an average size of 1.0 nm.

The overall count rate of 35 kcps was the lowest for any media. This count rate is very low compared to the others because there are no large proteins or particles in RPMI to scatter light (see Table 1).

Figure 22: RPMI Media - Volume Distribution

Fetal Bovine Serum

The size distributions for FBS and 10% FBS in RPMI, Figure 23 and Figure 24, were identical with a single population around 7 nm. FBS had a 10x higher count rate than the 10% FBS mixture, as expected. Albumin is the primary component of FBS, which has a diameter of 7.7 nm60. This is the most likely component responsible for the

DLS signal.

Figure 23: FBS Media - Volume Distribution

Figure 24: FBS 10% Media - Volume Distribution

X-Vivo 15 Serum Free Media

The serum free media had a size nearly identical to FBS. The distribution, Figure

25, had a single population at 6.5 nm. The count rate of 1.0x103 kcps was less than FBS, but significantly higher than the RPMI. The full contents of the media are not disclosed by the manufacturer, but it is interesting to note the similarity to FBS. Albumin is a major component of both FBS and serum free media, and has a diameter of 7.7 nm. As with the

FBS, albumin is the most likely component responsible for the DLS signal in the serum free media.

Figure 25: Serum Free Media - Volume Distribution

3.4.2 MPA Coated Quantum Dots

Quantum dot concentrations for all samples were 466 nM.

Water

The water sample, Figure 26, had an average size of 27 nm.

Figure 26: MPA Quantum Dots in Water - Volume Distribution

Roswell Park Memorial Institute Media

The RPMI sample, Figure 27, had an average size of 26 nm, which corresponds well to the 27 nm size of the quantum dots in water.

Figure 27: MPA Quantum Dots in RPMI - Volume Distribution

Fetal Bovine Serum

The distribution plots for FBS media alone and MPA coated quantum dots in FBS media, Figure 23 and Figure 28, looked similar. There were populations of 6.8 and 7.8 nm particles, respectively. The quantum dots in FBS media had an additional population of 57 nm particles. The 7.8 nm population was from FBS. The 57 nm population was from quantum dots. The 10% FBS sample, Figure 29, agrees with this explanation for the

FBS data. In this sample, the FBS concentration was a factor of 10 lower. The smaller population was no longer present, leaving only a population of 54 nm particles, similar to the 57 nm particles present in the FBS sample.

The increased size of the quantum dots in FBS media may be due to proteins binding to the quantum dots. The FBS media had a size of 7-9 nm; it could be supposed that FBS would increase the size of the quantum dots by 14-18 nm. Adding this to the 27 nm size of the quantum dots in water would give a size of 41-45 nm. This does not exactly match the 57 nm size measured. The difference may be particle aggregation.

Figure 28: MPA Quantum Dots in FBS - Volume Distribution

Figure 29: MPA Quantum Dots in FBS 10% Media - Volume Distribution

X-Vivo 15 Serum Free Media

The distribution plot for MPA coated quantum dots in serum free media, Figure

30, had populations at 12 nm and 39 nm. The 12 nm particles are likely from the serum free media, while the 39 nm particles are quantum dots. As with the FBS sample, the increased size relative to the quantum dots in water may be due to proteins associating with the particles, or aggregation.

Figure 30: MPA Quantum Dots in Serum Free Media - Volume Distribution

The MPA coated quantum dots did not agglomerate to large sizes in any of the media used in this experiment. RPMI had no effect on the particles, while FBS and serum free media increased their size either through protein association or small amounts of aggregation.

3.4.3 Thiocholine Coated Quantum Dots

Quantum dot concentrations for all samples were 466 nM.

Water

The distribution plot for thiocholine coated quantum dots in water, Figure 31, had one population of 17 nm particles.

Figure 31: Thiocholine Quantum Dots in Water - Volume Distribution

Roswell Park Memorial Institute Medium

Thiocholine coated quantum dots in RPMI media, Figure 32, had two populations of sizes, 20 and 62 nm. The 20 nm population corresponds to the 17 nm size of the quantum dots in water. The 62 nm population is made up of quantum dot aggregates. The increased ionic strength of RPMI compared to water destabilized the particles, as explained in the discussion of DLVO theory in Chapter 1.

Figure 32: Thiocholine Quantum Dots in RPMI - Volume Distribution

Fetal Bovine Serum

Thiocholine coated quantum dots in FBS, Figure 33, were similar to MPA coated quantum dots in FBS. There was a large population of 7 nm particles and a smaller population of 28 nm particles. The 7 nm particles are from the FBS media. The population at 28 nm is quantum dots. As with the MPA coated quantum dots, the larger size compared to particles in water may be due to protein association or aggregation.

Figure 33: Thiocholine Quantum Dots in FBS - Volume Distribution 52

The thiocholine coated quantum dots aggregated in the 10% FBS media (Figure

34). There were populations at 140 nm, 1200 nm, and a population that extended past the cutoff range of the instrument. The sample appeared cloudy.

Figure 34: Thiocholine Quantum Dots in FBS 10% Media - Volume Distribution

X-Vivo 15 Serum Free Media

The thiocholine coated quantum dots also aggregated in the serum free media

(Figure 35). Rather than having multiple populations as in the 10% FBS sample, the serum free media sample had one distribution at 3200 nm. The sample appeared cloudy.

Figure 35: Thiocholine Quantum Dots in Serum Free Media - Volume Distribution

Thiocholine coated quantum dots showed aggregation due to the ionic strength of the RPMI media. The particles did not aggregate in FBS, but did so in 10% FBS. The most significant aggregation occurred in serum free media.

3.5 Surface Charge in Biological Media

Table 2 below summarizes all zeta potential data for the biological media alone, positively charged quantum dots in the media, and MPA coated quantum dots in the media.

Media Alone Thiocholine QDs MPA QDs Media Zeta Count Zeta Count Zeta Count Potential Rate Potential Rate Potential Rate (mV) (kcps) (mv) (kcps) (mV) (kcps) RPMI 0 1 15 9.0x102 -34 1.4x102 RPMI/FBS -6 7 -8 2.6x104 -15 1.7x102 FBS -7 95 -10 1.4x104 -16 1.3x102 X-Vivo 15 -5 4 1 1.4x104 -24 1.5x102 Water - - 34 3.7x102 -44 2.2x102 Table 4: Summary of Zeta Potential Data

3.5.1 Media Only

Roswell Park Memorial Institute Media

As in the size data, RPMI media had the lowest count rate. It did not appear that any charged particles were observed. The phase plot, Figure 36, did not display any ordered phase shifting. The zeta potential distribution shown in Figure 37 had a very wide range of potentials, from -150 to 150 mV. This is due to the Zetasizer software attempting to fit noise. The minimum size for zeta potential measurements in the

Zetasizer Nano is 3.8 nm. The measured size for RPMI was 1 nm. The mean zeta potential given was 0 mV.

Figure 36: RPMI Media Phase Plot

Figure 37: RPMI Media Zeta Potential Distribution

Fetal Bovine Serum

The FBS media had the highest count rate of any media: 95 kcps. The phase plot in Figure 38 was not entirely random like the RPMI sample, but still contained considerable noise. The shape of the slow field reversal region (from 1.2 through 2.6 seconds) indicates negative particles were present. The zeta potential was -7 mV.

Figure 38: FBS Media - Phase Plot

Figure 39: FBS Media - Zeta Potential Distribution

The phase plot of the 10% FBS sample, Figure 40, was more dominated by noise than the FBS sample. The lower concentration was reflected in the count rates; 7 kcps for

10% FBS compared to 95 kcps for FBS. The distribution plot looked similar to RPMI in

Figure 37, indicating the data was too noisy to obtain a meaningful transform of the slow field data. In spite of this noise, a mean zeta potential was able to be determined from the fast field reversal data. The mean zeta potential was -6 mV for the 10% FBS.

Figure 40: FBS 10% Media - Phase Plot

Figure 41: FBS 10% Media - Zeta Potential Distribution

X-Vivo 15 Serum Free Media

The serum free media data in Figure 42 and Figure 43 were similar to the 10%

FBS sample. The count rate was low: 3.8 kcps. The phase plot was dominated by noise, again leading to a poor distribution plot. The fast field reversal was able to determine a mean zeta potential of -5 mV.

Figure 42: Serum Free Media - Phase Plot

Figure 43: Serum Free Media - Zeta Potential Distribution

3.5.2 MPA Coated Quantum Dots

Quantum dot concentrations for all samples were 466 nM.

Water

The phase plot of MPA coated quantum dots in water, Figure 44, demonstrates what good quality data looks like, in comparison to the phase plots for media-only samples. There was a visible pattern in the fast field reversal portion, and the slow field reversal portion showed a smooth noiseless slope. The mean zeta potential for this sample was -44 mV. The count rate was 220 kcps.

Figure 44: MPA Quantum Dots in Water - Phase Plot

Figure 45: MPA Quantum Dots in Water - Zeta Potential Distribution

Roswell Park Memorial Institute Media

The MPA coated quantum dots in RPMI also had plainly visible patterns in the phase plot, Figure 46. The mean zeta potential was -34 mV. The more neutral zeta potential of this sample was due to the increased ionic strength of the medium. Higher concentrations of counter-ions were present to shield the particles. The count rate of 135 kcps was significantly higher than RPMI’s 1 kcps.

Figure 46: MPA Quantum Dots in RPMI - Phase Plot

Figure 47: MPA Quantum Dots in RPMI - Zeta Potential Distribution

Fetal Bovine Serum

The phase plot of MPA coated quantum dots in FBS, Figure 48, was a clear improvement over FBS media alone, Figure 38. This indicates that quantum dots were being measured rather than the media. The mean zeta potential of -16 mV suggests that proteins from the media were associating with the particles and shielding their charge.

Figure 48: MPA Quantum Dots in FBS - Phase Plot

Figure 49: MPA Quantum Dots in FBS - Zeta Potential Distribution

The mean zeta potential of MPA coated quantum dots in 10% FBS was -15 mV, nearly the same as in FBS. The count rates were also nearly identical; 131 kcps for FBS and 168 kcps for 10% FBS.

Figure 50: MPA Quantum Dots in FBS 10% Media - Phase Plot

Figure 51: MPA Quantum Dots in FBS 10% Media - Zeta Potential Distribution

X-Vivo 15 Serum Free Media

The phase plot for MPA coated quantum dots in serum free media in Figure 52 looked similar to the particles in FBS. The mean zeta potential was -24 mV, again suggesting protein association. They were less negative than in water (-44 mV) but more negative than in FBS (-16 mV). The count rate of 148 kcps was similar to the count rates for MPA coated quantum dots in the other media.

Figure 52: MPA Quantum Dots in Serum Free Media - Phase Plot

Figure 53: MPA Quantum Dots in Serum Free Media - Zeta Potential Distribution

All of the media used in this experiment impacted the zeta potential of the MPA coated quantum dots. FBS had the strongest effect on the particles, changing their zeta potential from -44 mV to -15 mV.

3.5.3 Thiocholine Coated Quantum Dots

Quantum dot concentrations for all samples were 466 nM.

Water

The phase plot for thiocholine coated quantum dots in water, Figure 54, mirrored the MPA coated quantum dots in Figure 44. The slow field reversal showed a positive phase shift rather than negative. This indicated a positive charge, which was also seen in the zeta potential distribution in Figure 55. The zeta potential was 34 mV. The count rate of 367 kcps was slightly higher than for any of the negatively charged quantum dot samples, which ranged from 131 to 220 kcps.

Figure 54: Thiocholine QDs in Water - Phase Plot

Figure 55: Thiocholine QDs in Water - Zeta Potential Distribution

Roswell Park Memorial Institute Media

Similar to the MPA coated quantum dots, the magnitude of the zeta potential decreased when the thiocholine coated quantum dots were dispersed in RPMI media compared to water. The zeta potential went from 34 mV in water to 15 mV in RPMI. The higher count rate of 895 kcps indicates that aggregation occurred.

Figure 56: Thiocholine Quantum Dots in RPMI - Phase Plot

Figure 57: Thiocholine Quantum Dots in RPMI - Zeta Potential Distribution

Fetal Bovine Serum

The 13671 kcps count rate for thiocholine coated quantum dots in FBS was significantly higher than in water, suggesting aggregation. The phase plot and zeta potential distribution of thiocholine coated quantum dots in FBS, Figure 58 and Figure

59, look identical to the plots for FBS media alone in Figure 38 and Figure 39. The mean zeta potential was -10 mV, comparable to the -7 mV zeta potential of FBS media.

Figure 58: Thiocholine Quantum Dots in FBS - Phase Plot

Figure 59: Thiocholine Quantum Dots in FBS - Zeta Potential Distribution

Thiocholine coated quantum dots in 10% FBS, Figure 60 and Figure 61, look identical to the same particles in FBS. The mean zeta potential was -8 mV. The count rate was 26314 kcps, twice that of the particles in FBS. Increased count rates rule out the possibility that these are only measuring the FBS media rather than the quantum dots.

Larger aggregates would scatter light significantly more than the small species present in

FBS, so it can be assumed that the signal being measured is from those aggregates. The drastic change of the zeta potential from 34 mV in water to -10 mV in FBS, similar to the value for FBS alone, means that the charged species from FBS is strongly associating with the positively charged quantum dots.

Figure 60: Thiocholine Quantum Dots in FBS 10% Media - Phase Plot

Figure 61: Thiocholine Quantum Dots in FBS 10% Media - Zeta Potential Distribution

X-Vivo 15 Serum Free Media

The phase plot for thiocholine coated quantum dots in serum free media in Figure

62 appeared to have no signal at all, similar to Figure 42 for serum free media alone. The count rate of 14125 kcps indicates a significant amount of scattering from aggregated particles. The zeta potential distribution, Figure 63, appeared more ordered than the distribution for serum free media alone in Figure 43. The mean zeta potential was 1 mV, essentially neutral. As with the FBS sample, the count rate is high but the phase plot and mean zeta potential are essentially the same as for the media alone. A neutral species from the serum free media is strongly associated with the particles.

Figure 62: Thiocholine Quantum Dots in Serum Free Media - Phase Plot

Figure 63: Thiocholine Quantum Dots in Serum Free Media - Zeta Potential Distribution

The positive charge on these particles appeared to have a significant effect on their stability in biological media. Aggregation was seen in all media other than water, and in FBS and serum free media the charge changed entirely from positive to negative and neutral, respectively

3.6 Quantum Dot Association with Macrophages

Figure 64 summarizes flow cytometry experiments performed by Amber Nagy.

Parts A and B show the flow cytometry experiments for MPA coated quantum dots with and without Poly I, a scavenger receptor inhibitor. Parts C and D show the same experiments for thiocholine coated quantum dots. The experiments were all performed in

RPMI (left), RPMI+FBS (middle), and X-Vivo 15 serum free media (right), the same media that were the subject of discussion in the size and charge experiments done here.

As cells travel through a flow cytometer, they pass single file through the path of an excitation source. The fluorescence intensity is measured for each cell as it passes through. The plots in Figure 64 show the number of measurements (cells) on the y axis which emit a fluorescence intensity on the x axis. For example, in the top right plot, the red line for cells with no quantum dots shows that most of the cells have weak fluorescence intensity. This is expected, as there are no fluorescent quantum dots that could be associated with the cells. The purple line for cells cultured in a 250 nM quantum dot solution shows that the cells have much higher fluorescence intensity. The instrument is only measuring cells, so fluorescence is attributed to quantum dots that have associated with the cell.

Figure 64: Comparison of cell internalization of thiocholine and MPA coated quantum dots in various media 78

Observing the samples with no Poly I added, the effects of the media were determined. For MPA coated quantum dots, the RPMI sample showed the greatest amount of internalization, followed closely by the serum free media. The RPMI+FBS sample showed decreased internalization for all but the highest quantum dot concentration (250 nM). The media did not appear to have an effect on association of the thiocholine coated quantum dots with macrophages, though it was slightly increased for the RPMI sample compared to RPMI+FBS and serum free media.

As discussed in Chapter 1, Poly I associates with the LDLR/SR scavenger receptor pathway. Adding Poly I to a sample will block other particles from using this pathway. If the quantum dots enter the cell through this pathway, there should be decreased quantum dot association with the macrophages when Poly I is added. This clearly takes place for the MPA coated quantum dots in RPMI. The quantum dot uptake is lowered when Poly I is added. The uptake is not completely stopped, indicating that the

LDLR/SR pathway is not the only one that MPA coated quantum dots take into macrophages. Association is also decreased for MPA coated quantum dots in serum free media and RPMI/FBS. The thiocholine coated quantum dots are not affected by the addition of Poly I. The thiocholine coated quantum dots do not follow the LDLR/SR pathway into the macrophages.

Chapter 4: Discussion and Conclusions

4.1 Mechanism of CdSe/ZnS Microwave Synthesis

From these experiments, we propose a reaction mechanism for the microwave synthesis of CdSe/ZnS core-shell quantum dots. A population of small CdSe seed nanoparticles formed immediately upon mixing cadmium and selenium ions. These particles were protected from aggregation and further reactions by the thiol-linked protection of 3-MPA. As the microwave heating began, the particles were de-protected and underwent Ostwald ripening to form a monodisperse population of larger CdSe particles with an emission wavelength of approximately 550 nm. After 30 minutes, this growth was stopped by the addition of a ZnS cap, which began to form due to the release of sulfur from decomposing 3-MPA. This capping stopped the CdSe cores from continuing to grow into bulk material which would not fluoresce, and prevented trap-state defects from forming on the surface of the CdSe shell. As the ZnS shell continued to grow, the emission red-shifted and continued to increase in quantum yield until the ideal heating time of 80 minutes.

4.2 Optical Properties

The optical properties of these quantum dots matched closely with previous work utilizing this synthesis33, with some improvements. The absorption spectra in Figure 18 were typical of what would be seen in quantum dots. The absorption peaks near 525 nm were exciton peaks, corresponding to the particles’ band gaps. The peaks near 560 nm in

Figure 19, the emission spectra, were the main quantum dot emissions. The red shift of the emission wavelength from the absorption wavelength, called the Stokes shift, was expected, and is mostly attributed to interactions with the solvent61. The main peak had a small distribution (typically 40 nm full width half max). Because this wavelength is determined by band gap, and thus the size, of the quantum dot, the width of the main emission peak is also indicative of the mono or polydispersity of the particles. The low intensity broad peaks centered on 700 nm were from trap state emissions. Trap states can come from any number of possible deformities, making their distribution much wider.

Significant trap state emission is an indicator of poor quantum dot quality. If the surface is poorly formed or protected, the trap state emission will be increased, and will dominate the emission profile. This is not desirable, because the emission will be dimly spread across a wide range of wavelengths, rather than one strong color of emission. Quantum dots made during this research routinely had core emission:trap state ratios approaching

40-50.

4.3 Physical Properties of Quantum Dots in Water

The pH of the quantum dot solution had a larger effect on the MPA coated quantum dots (Figure 20) than the thiocholine coated quantum dots (Figure 21). This was expected due to the properties of the capping agents: MPA (Figure 65) has a negative charge because of a deprotonated carboxylic acid group. The pKa of MPA is 4.38. As the pH approached the pKa, more MPA molecules became protonated, making the overall zeta potential less negative. As the zeta potential increased, the quantum dots destabilized and began to aggregate. This began at pH 6.

Figure 65: 3-Mercaptopropionic Acid

In contrast, thiocholine (Figure 66) is a quaternary ammonium cation; it has a positive charge that is permanent and independent of pH. This is evident in Figure 21, where the zeta potential and size remained relatively unchanged across a wide pH range, from 7 to 2 pH units. The zeta potential decreased above pH 8. It is suspected that this was due to hydroxide ion interactions with the quantum dot surface. Hydroxide ions may bind to the quantum dots at open sites, particularly zinc or cadmium sites exposed due to shell defects. The thiocholine coated quantum dots were not stable above pH 9.

Figure 66: Thiocholine

4.4 Size of Quantum Dots in Biological Media

In previous work using this synthesis33, the particles were imaged using transmission electron microscopy and found to have a diameter of 5.0 nm. Our measured diameter for MPA quantum dots in water was 27 nm. Dynamic light scattering gives a hydrodynamic diameter: the diameter of the particle itself, any ligands attached to it, and solvent molecules which may be strongly associated with it. Intuitively, this should be larger than the bare particle radius which would be measured by other methods such as electron microscopy. While DLS does not give the exact diameter of a particle, it is a useful tool for characterizing colloidal suspensions.

A potential issue that arose during this experiment was interference from the media. Both the MPA and thiocholine coated quantum dots were overshadowed by the

FBS media in Figure 29 and Figure 34. It is important to analyze the media alone as well as the particles in the media to prevent mistaking particles from the media for the intended particles. A simple method of identifying the source of a population is to alter relative concentrations. This is well illustrated by thiocholine quantum dots in FBS and

10% FBS, Figure 33 and Figure 34. In FBS, there was a large population under 10 nm and a smaller population around 30 nm. When the concentration of FBS was reduced to

10%, the large population of particles under 10 nm disappeared, leaving only the

83 population around 30 nm. This is clear evidence that the smaller particles were from the

FBS media.

An indication that the media is interfering with the size determination of the intended particles is a relatively high count rate for the media alone. Ideally, the count rate should be significantly lower for the media than for quantum dots in the media. For example, RPMI media had 35 kcps, while the MPA and thiocholine capped quantum dots in RPMI were 4577 and 1702 kcps, respectively. The count rate increased significantly when quantum dots were added, so the signal can be assumed to be from quantum dots.

In the case of FBS, the count rate for the media alone was 39181, while MPA and thiocholine quantum dots were 48824 and 86249. In this case, the count rates are comparable, and some of the signal may be from the media.

4.5 Surface Charge of Quantum Dots in Biological Media

While zeta potential distribution plots are useful for quickly visualizing the data, they can be misleading. The plots tend to have very wide distributions, for instance in

Figure 57 for thiocholine quantum dots in RPMI. The distribution suggests that the solution has particles ranging from approximately -50 mV to +75 mV. It is unlikely that the solution contains particles at either of these extremes; rather there are particles central to the distribution which is broadened by Brownian motion.

For the media alone, only FBS gave data that can be considered reliable. It was the only media with a count rate higher than 10 kcps. RPMI had almost no count rate due to its small particle size. 3.8 nm is the listed minimum size particle for which the

Zetasizer instrument can measure a zeta potential, while RPMI media had a mean size of

1 nm. The Zetasizer did calculate a surface charge of -5 mV for the serum free media, but zeta potentials measured between positive and negative 5 are typically considered to be zero. Because we know that the 10% FBS sample should look similar to the FBS sample with lower signal, we can assume that the small negative drop in the right half of Figure

40 is real.

The MPA coated quantum dots did not aggregate in any of the media. This is evident in the consistent count rates between all of the samples, and was also seen in the size data. The surface charges do suggest, however, that the particles were affected by the media. None of the media samples were as negatively charged as the water sample. The

RPMI sample was slightly more positive, due to shielding from the high concentration of ions in RPMI, as explained in Chapter 1 using DLVO theory. Serum free media and the two FBS media had more significant effects on the quantum dots. The serum free media made the zeta potential more neutral: to -24 mV from -44 mV in water. Both FBS samples were increased even further to -16 and -15 mV. Proteins from the media, most likely albumin, associated with the quantum dots and decreased their effective surface charge. Albumin is known to interact with both positively and negatively charged nanoparticles55. Albumin is negatively charged, so it interacts electrostatically with the positively charged quantum dots. There are also sites on albumin which are known to bind to carboxylic acid groups.

The thiocholine coated quantum dots proved to be less stable in the media. The quantum dots as synthesized in water were 34 mV. While the particles were stable in

RPMI media, the zeta potential was lowered to 15 mV. This is the same effect seen with the negatively charged particles, due to the high ionic strength of the media. Interestingly, the FBS and 10% FBS media both made the thiocholine coated quantum dots negatively charged. The zeta potentials were comparable the values measured in the FBS media alone, indicating that the quantum dots were completely coated with negatively charged species from the media. As stated above, albumin is the most likely candidate. It has a negative charge, and is the most abundant protein in serum. Serum free media also coated the thiocholine coated quantum dots, but made them neutral. Because the formulation is proprietary, it is not clear what could be interacting with the quantum dots. It is possible that albumin was responsible, as suspected for the other samples, and that the aggregates were simply too large to measure the negative charge.

4.6 Quantum Dot Association with Macrophages

The results from the flow cytometry experiments can be compared to the data in

Table 3 and Table 4 to determine what impact size and charge have on quantum dot association with macrophages. MPA coated quantum dots in all three media had sizes below 100 nm. The magnitude of the surface charge increased in each media; -35 mV in

RPMI, -24 mV in serum free media, and -15 mV in RPMI/FBS. The association of MPA coated quantum dots with macrophages followed the same pattern; highest in RPMI, lower in serum free media, and lowest in RPMI/FBS. Thiocholine coated quantum dots were also less than 100 nm in RPMI, with populations of 20 and 62 nm particles. The charge of the thiocholine coated quantum dots in RPMI was +15 mV. These associated

86 with macrophages less than all of the MPA coated quantum dots. We propose from this data that quantum dot association with macrophages at this size is charge dependent, favoring negative charges. The thiocholine coated quantum dots were neutral and negative in serum free media and RPMI/FBS (1 mV and -8 mV), but still did not associate with macrophages. Both of these samples were aggregated (3200 and 1200 nm).

These particles were too large to follow the same uptake pathway as the non-aggregated

MPA coated quantum dots.

The addition of Poly I showed that small, negatively charged quantum dots follow the LDLR/SR uptake pathway. This is not the exclusive pathway they take into a cell, as there was still some association when it was blocked. Positively charged quantum dots, or large negative/neutral quantum dots, were less associated with the macrophages. Poly I had no effect on this association, so they do not follow the LDLR/SR pathway.

4.7 Comparison to Literature

The organic hot injection synthesis for quantum dots has been well characterized8.

Quantum dots are nucleated within seconds upon injection62. The solution is then cooled, after which a slow growth stage takes place. This leads to mono-disperse particles whose size can be easily chosen by quenching the slow growth phase.

For aqueous methods, the growth of II VI quantum dots has been found to follow

Ostwald ripening kinetics63. Recent in-situ XAFS experiments have confirmed that aqueous synthesis of II VI quantum dots follows the same fast nucleation-slow growth mechanism as the hot injection organic synthesis, with Ostwald ripening growth

87 kinetics64. These experiments did not observe two populations of particles as ours did, but they do support our conclusion that a small population of CdSe particles existed at the beginning of the reaction and dissolved through Ostwald ripening.

The size of aqueous synthesized quantum dots is typically controlled by the stabilizing ligand. It has been shown that using various thiols results in a variety of particle sizes9. After the fast nucleation, the thiol ligands bind to the quantum dots and prevent excessive growth. The consequences of this were seen when we attempted our synthesis without 3-MPA, after mixing the cadmium and selenium ions a flaky brown precipitate immediately formed. The red-shifting of the quantum dot emission as the ZnS shell grew has also been reported in literature59. Similar to this study, as the shell grew too thick, the quantum yield of our particles began to decrease.

The presence of bovine serum albumin in fetal bovine serum is well established57, as is its interaction with various nanoparticles both positive and negatively charged52,53,55,65,66. Albumin is known to affect the surface charge of nanoparticles, including making positive particles negative as we observed in our studies here55.

Our internalization studies agreed with the general patterns we have seen in other research. The MPA coated quantum dots strongly associated with the macrophages via a scavenger receptor pathway, which has also been observed in human epidermal keratinocytes38. This study found that neutral and positively charged quantum dots were poorly associated with the cells. We observed a general trend of less negative particles associating less with the macrophages. Positive and/or large nanoparticles are known to take different pathways into cells, and to associate less with them in general38,40,41.

4.8 Conclusions

In the microwave production of CdSe/ZnS core/shell quantum dots, we were able to determine a fast nucleation/slow Ostwald ripening growth mechanism. The disappearance of small particles and subsequent growth of larger particles was visualized via in-situ fluorescence spectroscopy for the first time.

The pH titrations demonstrate how a specific capping agent or charge could be utilized to control nanoparticle behavior. MPA and thiocholine capped quantum dots were both stable at near-neutral pHs. However, if the particle was expected to encounter acidic conditions, the thiocholine coated quantum dots would be more stable. If the particle was expected to encounter basic conditions, the MPA coated quantum dots would be more stable.

The capping agent used was also found to have an effect on the particle’s stability when exposed to complex mixtures. The size data showed that the MPA coated quantum dots were stable in all media. The zeta potentials, however, showed that the particles weren’t completely unaffected. The high ionic strength of RPMI media made the particles appear less negatively charged. The three media containing more complex mixtures of proteins (FBS, 10% FBS, and serum free media) all brought the particles’ zeta potentials much closer to zero. The media clearly interacted with the quantum dots and changed their properties.

For the thiocholine coated particles, the size data showed that the 10% FBS and serum free media samples aggregated while the others were stable. The thiocholine

89 coated quantum dots were stable in FBS and had a small particle size, but the zeta potential reversed, indicating association with a species in the media. The zeta potentials explain the instability of the 10% FBS and serum free media samples, as both bring the surface charge too close to zero for the particles to remain stable.

Albumin is the largest component in FBS and likely to be a large component in serum free media. It has been shown previously to interact with similar particles to those used here, so it is likely to be interacting with these particles as well. This does not, however, rule out that other components of these complex mixtures are also interacting.

The reversal of charge for the thiocholine coated quantum dots is a significant issue. In biological applications of quantum dots or other nanoparticles, they may be given a specific charge in order to control where they travel or how they behave under certain conditions. If the particle is coated once exposed to species similar to those in

FBS media, they may aggregate or switch charges. This would cause them to behave differently than desired. The media chosen in this experiment are all commonly used in biological experiments. The thiocholine quantum dots studied here would only be useful in water or RPMI.

Small negatively charged quantum dots were found to follow the LDLR/SR pathway into cells, among other routes. This association is charge dependent; more negatively charged particles were more strongly associated with the macrophages.

Positively charged quantum dots and large quantum dots did not follow the LDLR/SR pathway, and their uptake was not size or charge dependent.

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