110415 corrected thesis

Analysis of Quantum Dot Skin Penetration in aBarrier Compromised In Vivo Model

by

Luke Jonathan Mortensen

Submitted in Partial Fulfillmentof the

Requirements for the Degree

Doctor of Philosophy

Supervised by

Professor Lisa A. DeLouise

Department of Biomedical Engineering

Arts, Sciences and Engineering

Edmund A. Hajim School of Engineering and Applied Sciences

University of Rochester

Rochester, New York

2011

ii

Dedication

I would like to dedicate this thesis to my beautiful loving wife Jenny, whose love

and understanding helped me be free to make this effort what it is today.

“The wind bloweth whence it listeth”

iii

Curriculum Vitae

Luke J. Mortensen was born in Wauseon, Ohio on June 16, 1982. He attended the

University of Toledo (Toledo, Ohio) from 2000-2003 and graduated with a Bachelor

of Science in Biomedical Engineering and a minor in Biological Chemistry. He

enrolled in the Biomedical Engineering Department at University of Rochester in

2005 and received a Master of Science degree in 2006. He has pursued research

in the fields of nanotechnology, skin permeability, near-IR tissue imaging, and

nanotoxicology in the lab of his advisor Professor Lisa A. DeLouise.

iv

Acknowledgements

I would like to thank my thesis advisor, Prof. Lisa DeLouise, who has helped me

to learn to strive for my potential as a researcher and has always reminded me of

the value of an inquisitive mind. She has been instrumental in my development as

a scientist and an engineer, and I will always remember her guidance and support

over the years. Prof. Benjamin Miller has been another invaluable member of

my committee and acting second advisor. His intensity, perspective, and encour-

agement have played a major role in shaping my vision of what a scientist can

aspire to become. Prof. Jim Zavislan has also provided constant support and

opinion over the years, helping me to learn the ropes of the optics field and offer-

ing his unique views and insight into experimental and life questions. Prof. Alice

Pentland has been of great assistance, and her advice on refining experiments and

knowledge of dermatology has helped shape my project over the years. All of my

committee members have played a major role in my project directions and the

character of my scientific thought, and for that I am greatly appreciative.

I would also like to thank the collaborators who made this thesis possible. I

would like to thank Prof. Alison Elder for her assistance in experimental planning

on the organ analysis studies and Bob Gelein for running the atomic absorption

analysis. Additionally, Karen Bentley and Gayle Schneider at the TEM core

have made all the TEM data in my thesis a possibility and been great to talk

ACKNOWLEDGEMENTS v

to and exceptionally patient with me during the past years. Karen Vanderbilt

has been key in performing incredible cryo-sectioning for me when I needed it.

Joanne VanBuskirk and Fatat Sleiman have been critical in teaching me how to

perform animal studies and other laboratory techniques over the years. Dr. Anna

DeBenedetto and Dr. Lisa Beck have been of great assistance in experimental

discussions and TEWL measurements. Chris Evans in the lab of Todd Krauss

has been a great help with QD chemistry discussions and keeping me stocked

with QDs. Chris Glazowski has been a great friend who has been instrumental in

the planning and building of the confocal microscope, and without his help that

portion of my thesis would not have been possible.

I would like to thank the department administrators in the Departments of

Biomedical Engineering and Dermatology, especially Donna Porcelli, Stefanie

D’Orazio, Fran Parrish, Jayne Kresinske, and Carol Pierce.

I would like acknowledge the friendship and support of my extended lab family.

My fellow full time members in the DeLouise lab- Ut-Binh Giang, Hong Zheng,

Siddarth Chandrasekaran, Christine Suss, and Meghan Jones have all made the

experience at the University of Rochester something special and their input on

presentations and experiments is a big part of where my research is today. I would

like to thank in particular the students that I mentored, Supriya Ravichandran

and Renea Faulknor, for helping me to grow. Supriya has been an incredible help

and friend over the years, and lively discussions about the tortoise will always

bring a smile. Renea was undergraduate researcher who is pursuing her PhD at

Rutgers, and her consistency and growth over her busy years here set the bar high.

My cubicle mates Alon Mantel and Lisa Bonanno have provided plenty of lively

discussion on science and life over the years and will be fondly remembered. Lisa

B. has been a good friend, and no matter where she and Jon end up, I’m sure our

ACKNOWLEDGEMENTS vi

friendship and collaboration will continue. Hsin-I Peng has been a good friend

and great to bounce ideas off of during the postdoc application process, and I wish

her luck as she moves on to a new postdoc as well. The other members of Ben

Miller’s lab- Leslie Ofori, Amrita Yadev, Rashmi Sriram, and Mark Lifson- with

whom we collaborate closely have always provided important insight and advice

during my graduate school career and they are certainly appreciated.

I would like to especially thank my family for their support and encouragement

over the years. My parents Rob and Sharon have instilled in me the drive to learn

and understand the world around me and for that I am thankful. My siblings Jesse,

Katie, and Lydia have all been very supportive and encouraging of my thesis work.

My Grandmother Bernita Jess has been a great inspiration as she has continued

to learn and grow even into her golden years. Of course, the nearest and dearest

place in my heart is reserved for my wife Jenny. Her support and discussion have

made me a better engineer, thinker, and person, and she has provided constant

encouragement and love. For this I will always be grateful.

Finally, I would like to acknowledge funding sources. This work was supported

by the National Science Foundation (CBET 0837891) and the National Institute

of Health (NIDA K25AI060884).

vii

Abstract

Exposure to engineered nanoparticles (NPs) is becoming near-inescapable as their unique

size dependent properties have ensured integration into a wide range of consumer prod-

ucts and research tools. One of the most biologically impactful of these applications is

in consumer products such as sunscreens and other cosmetics, where consumer use com-

monly consists of topical application to UVB damaged skin. A number of studies have

investigated the ability of NPs to penetrate the skin barrier, but very few are available

on NP permeation differences that result from clinically relevant skin barrier disrup-

tions such as UVB. This thesis uses semiconductor quantum dots (QDs) as a model

nanoparticle to investigate the impact of UVB on skin permeability. QDs are NPs with

advantageous fluorescence properties including high quantum yield, broad excitability,

and narrow emission bandwidth. This doctoral dissertation evaluates the impact of UVB

on QD skin penetration, investigates the effects of UVB primary keratinocyte QD cel-

lular interaction, and expands the technical palette with whole tissue confocal imaging

development.

UVB radiation causes a host of biological changes in the skin, one of which is epi-

dermal barrier disruption. In the first portion of this thesis, the skin penetration of

carboxylated QD through the skin of SKH-1 mice with and without UVB exposure was

evaluated immediately after irradiation. Skin samples collected at 8 and 24 hours after

QD application demonstrated low levels of penetration in non-UVB exposed mice and

qualitatively higher but still low levels of QD penetration in the UVB exposed mice. To

ABSTRACT viii

approach a quantitative evaluation of both UVB-induced defect and QD skin penetra-

tion, this dissertation next used a designed experiment approach to evaluate the effects

of UVB on skin barrier function as measured by transepidermal water loss and the im-

pact of UVB on skin penetration of QDs with atomic absorption spectroscopy. UVB

induced a strong defect that peaked 4-6 days after exposure. Carboxylic acid coated

QDs were applied to SKH-1 mice over the peak barrier disruption 24 hours, and both

a qualitative increase in skin penetration after UVB exposure using microscopy and a

low-level quantitative increase in Cd levels in the liver were found, suggesting increased

systemic access. Interestingly, experiments found statistically significant but still low

levels of QD collection in the lymph nodes without UVB exposure whose magnitude

decreased with UVB.

Increased QD skin penetration with UVB exposure suggests that topically applied

QDs will be able to interact with local cells in the epidermis. To investigate this pos-

sibility, differences in acute QD cytotoxicity and uptake of carboxylated QDs between

proliferative and differentiated primary keratinocytes with and without UVB exposure

have been evaluated. Despite similarities between proliferative and differentiated ker-

atinocytes in UVB and QD cytotoxicity, the proliferative cells have a much greater ability

to endocytose QDs than differentiated cells. These results suggest the greater potential

for QD interaction with proliferative basal and suprabasal cells in the epidermis which

could potentiate a higher risk of possible long-term effects from NP contact with UVB

exposed skin.

A challenge in the evaluation of NP skin penetration is sampling error and other

problems associated with histological processing. Current literature suggests the useful-

ness of confocal or multiphoton microscopies to address these issues. The final portion of

this thesis introduces the design, implementation, and validation of a fluorescence and

reflectance confocal microscopy system that utilizes far-red excitation to detect near-

IR lead sulfide QDs through ex vivo human stratum corneum and in the epidermis.

ABSTRACT ix

The tested system achieves QD sensitivity measures on par with those reported in the

literature for other techniques, and is demonstrated to detect QDs permeating skin.

This dissertation presents the first published results to evaluate the impact of UVB

on skin penetration of QDs and keratinocyte interaction with QDs, and has advanced

technology for whole-tissue confocal microscopic evaluation of QD skin penetration. Im-

portant advancements have been made, suggesting that UVB may increase risk of sys-

temic exposure to NPs, and that the lymphatic system may play an important role in

the translocation of topically applied NPs.

x

Table of Contents

Title Page i

Dedication ii

Curriculum Vitae iii

Acknowledgements iv

Abstract vii

Table of Contents x

List of Abbreviations xv

List of Tables xvii

List of Figures xviii

Foreword 1

1 Introduction 3

1.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.2 Quantum Dot Nanoparticles . . . . . . . . . . . . . . . . . . . . . 6

TABLE OF CONTENTS xi

1.3 The Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.3.1 Skin Structure . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.3.2 Acute Injury vs. UVB Barrier Disruption Mechanisms . . 13

1.4 The Nano-Bio Interaction . . . . . . . . . . . . . . . . . . . . . . 15

1.4.1 NP Skin Penetration . . . . . . . . . . . . . . . . . . . . . 15

1.4.2 QD Interaction With the Local Epidermal Cell Types . . . 20

1.4.3 Systemic Impact . . . . . . . . . . . . . . . . . . . . . . . 23

1.5 Evaluation of Skin Barrier Function . . . . . . . . . . . . . . . . . 25

1.6 Evaluation of In Vivo QD Skin Penetration . . . . . . . . . . . . . 27

1.6.1 Histological Sectioning . . . . . . . . . . . . . . . . . . . . 29

1.6.2 Transmission Electron Microscopy . . . . . . . . . . . . . . 30

1.6.3 Elemental Analysis . . . . . . . . . . . . . . . . . . . . . . 32

1.6.4 Advanced Microscopy . . . . . . . . . . . . . . . . . . . . . 33

1.7 Statistical Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . 37

1.8 Thesis Organization . . . . . . . . . . . . . . . . . . . . . . . . . . 39

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2 UVB and Immediate QD Skin Permeability 65

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

2.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . 69

2.2.1 QDs and Vehicle Preparation . . . . . . . . . . . . . . . . 69

2.2.2 QD Application to Mice . . . . . . . . . . . . . . . . . . . 70

2.2.3 UVB Radiation Protocol . . . . . . . . . . . . . . . . . . . 71

2.2.4 Skin Tissue Cryo-Processing . . . . . . . . . . . . . . . . . 71

2.2.5 Confocal Microscopy . . . . . . . . . . . . . . . . . . . . . 72

2.2.6 Transmission Electron Microscopy . . . . . . . . . . . . . . 74

TABLE OF CONTENTS xii

2.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . 75

2.3.1 Application Vehicle Characterization . . . . . . . . . . . . 75

2.3.2 Validation of UVB Induced Skin Response . . . . . . . . . 77

2.3.3 QD Penetration: Effect of UVB . . . . . . . . . . . . . . . 79

2.3.4 QD Penetration: Mechanistic Insight . . . . . . . . . . . . 83

2.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

3 UVB and Delayed QD Skin Permeability 96

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

3.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . 100

3.2.1 QD Functionalization and Vehicle Preparation . . . . . . 100

3.2.2 UVB Irradiation and TEWL . . . . . . . . . . . . . . . . 101

3.2.3 QD Application to Mice . . . . . . . . . . . . . . . . . . . 102

3.2.4 Dissection and Organ Analysis . . . . . . . . . . . . . . . . 102

3.2.5 Skin Tissue Cryo-Processing . . . . . . . . . . . . . . . . 103

3.2.6 Transmission Electron Microscopy . . . . . . . . . . . . . 104

3.2.7 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . 105

3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

3.3.1 TEWL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

3.3.2 QD Characterization . . . . . . . . . . . . . . . . . . . . . 109

3.3.3 Fluorescence Microscopy Evaluation of QD Skin Collection

and Penetration . . . . . . . . . . . . . . . . . . . . . . . 109

3.3.4 Ultrastructural TEM Analysis of Penetration Pathways . . 114

3.3.5 Quantitative Distal Organ Analysis . . . . . . . . . . . . . 115

3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

TABLE OF CONTENTS xiii

3.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

4 UVB, QDs, and Primary Keratinocytes 137

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

4.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . 141

4.2.1 QD Functionalization . . . . . . . . . . . . . . . . . . . . 141

4.2.2 Primary Keratinocyte Isolation, Culture, and Differentiation 142

4.2.3 UVB Irradiation . . . . . . . . . . . . . . . . . . . . . . . 143

4.2.4 QD Application . . . . . . . . . . . . . . . . . . . . . . . . 144

4.2.5 Flow Cytometric Analysis . . . . . . . . . . . . . . . . . . 144

4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

4.3.1 Cellular Toxicity of UVB . . . . . . . . . . . . . . . . . . . 145

4.3.2 Cellular Toxicity of QDs . . . . . . . . . . . . . . . . . . . 147

4.3.3 Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . 148

4.3.4 Flow Cytometry and QD Uptake . . . . . . . . . . . . . . 150

4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

4.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

5 Near-IR QD Confocal Imaging 169

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

5.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . 172

5.2.1 Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . 172

5.2.2 Estimation of Sensitivity . . . . . . . . . . . . . . . . . . . 175

5.2.3 Quantum Dot Imaging . . . . . . . . . . . . . . . . . . . . 180

5.2.4 Skin preparation . . . . . . . . . . . . . . . . . . . . . . . 181

TABLE OF CONTENTS xiv

5.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . 183

5.3.1 Sensitivity Estimates . . . . . . . . . . . . . . . . . . . . . 183

5.3.2 Experimental Validation and Model Comparison . . . . . . 186

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

6 Conclusions and Future Directions 202

A Chapter 2 Supplementary Data 208

B Chapter 3 Supplementary Data 215

xv

List of Abbreviations

ANOM Analysis of Means

ANOVA Analysis of Variance

Cd Cadmium

Diam. Diameter

EGFR Epidermal Growth Factor Receptor

KGFR Keratinocyte Growth Factor Receptor

LOD Limit of Detection

LOQ Limit of Quantification

LUT Look Up Table

MPE Maximum Permissible Exposure

N.Q. Below the Limit of Quantification

Near-IR Near-Infrared

NPs Nanoparticles

PEG Polyethylene Glycol

LIST OF ABBREVIATIONS xvi

QDs Quantum Dots

ROS Reactive Oxygen Species

SHG Second Harmonic Generation

TEM Transmission Electron Microscopy

TEWL Transepidermal Water Loss

TiO2 Titanium Dioxide

UVA Ultraviolet A Radiation

UVB Ultraviolet B Radiation

UVR Ultraviolet Radiation

ZnO Zinc Oxide

xvii

List of Tables

1.1 Common Zα and Zβ Values . . . . . . . . . . . . . . . . . . . . . 38

3.1 Cd Organ Accumulation . . . . . . . . . . . . . . . . . . . . . . . 117

xviii

List of Figures

1.1 QD Absorbance and Emission Spectra . . . . . . . . . . . . . . . 9

1.2 Skin Architecture . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.3 UVB Induced TEWL Defect . . . . . . . . . . . . . . . . . . . . . 28

1.4 Visible-Range Tissue Autofluorescence . . . . . . . . . . . . . . . 31

1.5 Distal Organ Location . . . . . . . . . . . . . . . . . . . . . . . . 34

2.1 QD Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

2.2 Stratum Corneum Thickening . . . . . . . . . . . . . . . . . . . . 76

2.3 QD Collection Trends . . . . . . . . . . . . . . . . . . . . . . . . . 78

2.4 Hair Follicle QD Collection . . . . . . . . . . . . . . . . . . . . . . 80

2.5 UVB Induced QD Skin Penetration . . . . . . . . . . . . . . . . . 82

2.6 QD Penetration Pathway . . . . . . . . . . . . . . . . . . . . . . . 84

2.7 QD EDAX Confirmation . . . . . . . . . . . . . . . . . . . . . . . 86

3.1 Impact of UVB on TEWL . . . . . . . . . . . . . . . . . . . . . . 106

3.2 QD Characterization . . . . . . . . . . . . . . . . . . . . . . . . . 110

3.3 Microscopy of Histological Sections . . . . . . . . . . . . . . . . . 113

3.4 Control TEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

3.5 QD Skin Penetration by TEM . . . . . . . . . . . . . . . . . . . . 119

3.6 Elemental Organ Analysis . . . . . . . . . . . . . . . . . . . . . . 122

LIST OF FIGURES xix

4.1 Primary Keratinocyte UVB Cytotoxicity . . . . . . . . . . . . . . 146

4.2 Primary Keratinocyte QD Cytotoxicity . . . . . . . . . . . . . . . 149

4.3 Fluorescence Microscopy of Cellular Uptake . . . . . . . . . . . . 151

4.4 Primary Keratinocyte Gating Scheme . . . . . . . . . . . . . . . . 153

4.5 Primary Keratinocyte QD Uptake . . . . . . . . . . . . . . . . . . 156

5.1 System Schematic . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

5.2 Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . 182

5.3 Model QD Responsivity . . . . . . . . . . . . . . . . . . . . . . . 184

5.4 Experimental QD Responsivity . . . . . . . . . . . . . . . . . . . 187

5.5 Model and Experimental System Responsivity . . . . . . . . . . . 189

5.6 QD Skin Penetration . . . . . . . . . . . . . . . . . . . . . . . . . 191

A.1 Malvern Raw Particle Size . . . . . . . . . . . . . . . . . . . . . . 209

A.2 Epidermal Thickness Post-UVB . . . . . . . . . . . . . . . . . . . 210

A.3 Thickened Skin 4.5 Days Post-UVB . . . . . . . . . . . . . . . . . 211

A.4 QD Collection in Defects . . . . . . . . . . . . . . . . . . . . . . . 212

A.5 Silver Enhanced Particle Size . . . . . . . . . . . . . . . . . . . . 213

A.6 Negative Control TEM and EDAX . . . . . . . . . . . . . . . . . 214

B.1 ANOM Chart for TEWL . . . . . . . . . . . . . . . . . . . . . . . 216

B.2 UVB Peak Dose Morphology . . . . . . . . . . . . . . . . . . . . . 217

B.3 Back Measurement Location . . . . . . . . . . . . . . . . . . . . . 218

B.4 TEM Negative Control . . . . . . . . . . . . . . . . . . . . . . . . 219

1

Foreword

Portions of the research discussed in this thesis have been presented in previ-

ously published form. In Chapter 1, sections were adapted from DeLouise, L.A.,

Mortensen, L.J., and Elder, A. Breaching epithelial barriers – physiochemical fac-

tors impacting nanomaterial translocation and toxicity, in Safety of Nanoparticles,

Ed. Webster, T.J. 2008 Springer, New York. This book chapter was co-authored

by Prof. Alison Elder and Prof. Lisa DeLouise, but I was primary author on

the sections adapted. Additionally, sections were adapted from Mortensen, L.J.,

Ravichandran, S., Zheng, H., and DeLouise, L.A. Progress and challenges in quan-

tifying skin permeability to nanoparticles using a quantum dot model, J Biomed

Nanotechnol 2010, 6, 596. This manuscript was co-authored with Prof. Lisa

DeLouise and experimental support provided by Supriya Ravichandran and Hong

Zheng. Portions of Chapter 2 were adapted from Mortensen, L.J., Oberdörster,

G., Pentland, A.P., and DeLouise, L.A. In vivo skin penetration of quantum dot

nanoparticles in the murine model: the effect of UVR, Nano Lett 2010, 8, 2779, an

article that was co-authored with Prof. Lisa DeLouise, Prof. Gunter Oberdörster,

and Prof. Alice Pentland. Sections of Chapter 3 were adapted from Mortensen,

L.J., Gelein, R., Bentley, K.L.D.M., Elder, A., and DeLouise, L.A. Steps towards

quantification of quantum dot skin penetration risk with UVB barrier impair-

ment as measured by transepidermal water loss, a manuscript submitted to Tox-

FOREWORD 2

icol Sci and coauthored by Prof. Lisa DeLouise and Prof. Alison Elder. Atomic

absorbance spectroscopy was executed by Robert Gelein, and TEM assistance

provided by Karen Bentley. Data in Chapter 4 was adapted from Mortensen,

L.J., Ravichandran, S., and DeLouise, L.A. The impact of UVB and differenti-

ation state on quantum dot interactions with primary keratinocytes, submitted

to Nanotoxicology 2011, which manuscript was co-authored with Prof. Lisa De-

Louise. Primary keratinocyte experiments were performed in collaboration with

Supriya Ravichandran. Sections of Chapter 5 were adapted from Mortensen, L.J.,

Glazowski, C.E., Zavislan, J.M., and DeLouise, L.A. Near-IR fluorescence and

reflectance confocal microscopy for imaging of quantum dots in mammalian skin,

a manuscript submitted to Biomedical Optics Express that was co-authored with

Prof. Lisa DeLouise and Prof. James Zavislan. Confocal microscope planning

and execution was performed in collaboration with Chris Glazowski. PDMS mi-

crowells used in this section were made by Ut-Binh Giang.

3

Chapter 1

Introduction

Quantum dot nanoparticles, biological in-teractions, and evaluation techniques 1

1Portions of this chapter are adapted from: a) DeLouise, L. A., Mortensen, L. J., and Elder,A. Breaching Epithelial Barriers – Physiochemical Factors Impacting Nanomaterial Transloca-tion and Toxicity, in Safety of Nanoparticles. Ed. Webster, T. J. 2008, Springer, New York. andb) Mortensen, L. J., Ravichandran, S., Zheng, H., and DeLouise, L. A. Progress and Challengesin Quantifying Skin Permeability to Nanoparticles Using a Quantum Dot Model, J. Biomed.Nanotechnol. 2010, 6, 596.

CHAPTER 1. INTRODUCTION 4

1.1 Motivation

The continued success of the nanotechnology field has meant an ever greater

chance for nanoparticle (NP) human contact. Over the last 20 years, a large

number of NP containing products have been introduced to consumers including

cosmetics containing nano-TiO2 and –ZnO sunscreens. More recently, products in-

cluding quantum dot (QD) LED’s, carbon nanotube containing sports equipment,

nanosilver impregnated antimicrobials, and water purification technologies have

come to market. [1,2] In addition to consumer products, many potential biomedi-

cal applications leveraging NP properties for fluorescent imaging, drug delivery,

and MRI contrast enhancement are in developmental stages. [3–7] Their increasing

presence in research and manufacturing fields means an increased risk for human

exposure, and concurrent research has investigated the interaction between NPs

and major human exposure routes: the gastrointestinal tract, [8,9] the lungs, [10–12]

and the skin. [13–15]

The concept of NPs was first suggested by Richard Feynman in his oft-quoted

lecture “There’s plenty of room at the bottom” in 1959. [16] As his concept so-

lidified into a scientific field, the usual guideline for a NP became an engineered

material with one dimension <100 nm. NPs are useful due to unique size de-

pendent properties not present in bulk materials born of quantum confinement

or surface area to volume ratio effects that allow a wide range of advantageous

physical, optical, and chemical properties. Cosmetics such as ultraviolet radiation

(UVR) protective sunscreens (which often contain ZnO and TiO2 NPs) are one

of the applications with the highest opportunity for human system interaction,

in particular with the skin. Since UVR has a long been known to have a barrier

impairing effect, [17] frequent use of sunscreens is likely to occur on sun-damaged


skin where penetration is more likely. Moreover, there is little standardization in

the NP physicochemical properties (e.g. size and surface characteristics) or vehicle

used in sunscreen formulation. These characteristics may greatly impact NP skin

penetration risk. However, when the skin barrier is intact minimal penetration

is observed for the NP-sized TiO2 and ZnO commonly used in sunscreens. [18–25]

Similar results have been found with intact skin barrier for hydrophilic polymer

NPs, [26] other metal NPs, [27,28] and quantum dots (QDs). [13–15,29] However, when

subjected to a disrupting agent such as chemical penetration enhancers with ZnO

NPs, [30] tape stripping with QDs, [31] dermal abrasion with QDs, [15,29] sonophoresis

with QDs and gold NPs, [32–34] or UVB exposure with QDs [14,35] the skin barrier

may become more permeable to NPs. These studies, including work to be pre-

sented in this doctoral thesis, suggest that depending on the severity of insult

barrier compromised skin can be breached by NPs, and are able to collect sys-

temically [36] and interact with the local cellular environment in a potentially toxic

fashion. [37–40] Given the common occurrence of NPs in sunscreens used in combi-

nation with UVB exposure, and the known ability of UVB to induce skin barrier

damage, it is important to determine the effects of UVB on skin penetration of

NPs and NP interaction with the local cellular environment- the focus of this

thesis.

This doctoral thesis focuses on the skin penetration of hard insoluble NPs

such as titanium dioxide and quantum dots. One of the primary challenges of

testing skin penetration of metal oxide NPs is an inability to accurately deter-

mine specific NP localization in the skin, such as to the stratum granulosum or

basal keratinocytes. To overcome this problem, fluorescent quantum dots (QD)

provide an advantage in detection. QD have broad excitability, a narrow emis-

sion bandwidth, high quantum yield, and easily modified surface properties. [41]


They are of great interest in the life sciences as targeted probes to examine bio-

logical processes, cell features, and even potentially medical diagnostics and drug

delivery. [4] Due to the relative similarity of QD and metal oxide NP size, shape,

mechanical properties, and surface characteristics in the raw material forms (often

used for skin penetration studies), scientists commonly use them as a model NP

for skin penetration studies. [13,15,29,34,42,43]

This thesis investigates three primary areas: 1. The impact of UVB on skin

barrier function as measured by transepidermal water loss (TEWL) and its cor-

relation to the skin penetration of QDs, 2. The impact of UVB and primary

keratinocyte differentiation on QD interaction in vivo, and 3. Development of a

confocal microscope designed to enable whole tissue evaluation of the penetration

of near-IR QDs. In the introduction, the background of the field and impor-

tant technical elements will be elaborated, thereby illuminating the literature and

technical underpinnings of the research to follow.

1.2 Quantum Dot Nanoparticles

QDs are a major class of NPs (particles with one dimension <100 nm) that

have become popular in a number of research fields due to size dependent quan-

tum confinement effects. These quantum confinement effects can be exploited to

provide fluorescent probes with broad excitability, a narrow emission bandwidth,

high quantum yield, and easily modified surface properties. [41] They are of great

interest in the life sciences for use as targeted biological probes, medical diagnos-

tic tools, and cancer therapeutics. [4,44,45] In addition, they have shown promise as

light conversion agents in solar cells, quantum information containing packets for

quantum computing, and light emitting diodes.


QDs are normally synthesized in a colloidal phase with the size and shape

controlled by organo-metallic semiconductor substrate, the temperature, and the

surfactant (commonly trioctyl phosphine/trioctyl phosphine oxide). [46] The emis-

sion frequency is modified by the size of the QD, which is controlled in turn by

the reaction time. QDs are synthesized in organic solvents, so to be used in

the aqueous biological environment they must be transferred into water by way

of surface modification. The primary strategies used for surface modification of

QDs are ligand exchange, which replaces the organic ligands with water soluble

ligands, and amphiphilic ligand encapsulation, which covers the organic surface

ligands with amphiphilic polymers that engage in hydrophobic interactions on one

side of the molecule and interacts with the surrounding aqueous solution on the

other. The new hydrophilic ligands affect the way that the resulting water-soluble

QD interact with their surrounding environment and can be used as attachment

sites for targeting molecules. Ligand exchange substitutes the organic capping

molecules with hydrophilic molecules that usually have a terminal thiol or hy-

drophobic region to coordinate with atoms on the semiconductor QD surface and

an opposing hydrophilic end group. Some examples of surface chemistries that

are prepared in this fashion are mercaptoundecanoic acid, [47] thiol-PEG, [3] cys-

teine, [48] and dihydrolipoic acid. [49] Dihydrolipoic acid is a commonly used ligand

due to its bi-dentate thiol linker that yields stable QDs with a strong negative

charge. [3,49–51] One of the features about QDs that sets them apart from tradi-

tional molecular dyes is their absorbance profile that increase with higher energy

light past the first exciton (Figure 1.1). This feature allows the usage of one

laser source to excite multiple emission wavelengths of QDs, and increases ease in

multiplexing fluorescent assays. Additionally, the intrinsic fluorescence that can

be tuned to the near-IR using alternative core compositions such as InAs, PbS or


PbSe allows QDs to operate in a regime that allows much better fluorescent detec-

tion in deep tissue, and could be of great use in whole body imaging, metastatic

tumor identification and observation, and detection of QD skin penetration.

1.3 The Skin

1.3.1 Skin Structure

The skin is the largest organ in mammals, and comprises one of the primary

interfaces with the outside world, along with the gastrointestinal and respiratory

tracts. It provides many functions, the most important of which is regulation of

the interface between the exterior world and the internal environment. Skin has

evolved a multilayer physical architecture comprised of the innermost subcutis, the

dermis and the outermost epidermis and stratum corneum. Each layer consists of

different cell types, biomolecules, and appendages that uniquely work together to

maintain the barrier function.

The subcutis and the dermis are the innermost portions of the skin. They

both provide nutrient and waste transport for the epidermis along with mechani-

cal protection and thermal insulation. The dermis shares structural features with

the subcutis, such as the presence of adipose tissue, collagen-glycosaminoglycan

complexes, loose connective tissue, and elastic proteins. Macrophages are an im-

portant cell type in the dermis that phagocytize any foreign substances that might

be present in the tissue, even polymer microspheres with diameter 0.5-3.5 µm di-

ameter, and degrade them with lysosomes. [28] A variety of skin appendages reside

in the dermis including hair follicles, nerve endings, and secretory glands (sweat,

sebaceous, and apocrine). Glands provide a supporting role in preventing skin

penetration by releasing lipids to keep the skin flexible and hydrated and salty


0

0.2

0.4

0.6

0.8

1

520 540 560 580 600 620 640 660 680 700

% M

ax

Wavelength (nm)

Figure 1.1: QD Absorbance and Emission SpectraThe absorbance and emission profile for a DHLA functionalized QD with an emis-sion peak of 620 nm. For the CdSe/ZnS QD with a 620 nm peak emission depicted,the Stokes shift is around 20 nm.


aqueous solutions (sweat) to rinse off substances and salt them out of solution.

Hair follicles may allow penetration access to the dermis that is otherwise blocked

off by the stratum corneum. The hair follicle is constructed with a hair sur-

rounded by several layers of lining. The inner root sheath, outer root sheath, and

fibrous sheath all serve to prevent the entrance of outside materials through the

hair follicle, but the hair follicle barrier is not as fully developed as the stratum

corneum. This weakness has been confirmed in studies showing hair follicles to

be prime targets for NP skin penetration and drug delivery. [52,53] The proximity

of vasculature, the presence of follicular stem cells, and the presence of antigen

presenting Langerhans cell make hair follicle permeability an important possibil-

ity. Langerhans cells in particular are antigen presenting cells that are known to

migrate in response to barrier disruption, [54] and have been demonstrated to up-

take NPs that penetrate the skin. [55] This property has been exploited with a gene

gun delivery of DNA vectors containing polymer NPs that enable the tracking of

Langerhans cells, and suggest use in vaccines and drug delivery. [56]

The basement membrane separates the dermis and the epidermis. It consists

of closely packed collagen IV, laminin, and fibronectin. The basement membrane

is bordered on the apical side by the epidermis, which consists of several layers in-

cluding the stratum basale, stratum spinosum, stratum granulosum, and stratum

corneum. The stratum basale is made up of keratinocytes (∼80%), which are re-

sponsible for maintaining the proliferative potential of the skin, and melanocytes,

which provide pigment. Keratinocytes are the majority cell in the epidermis,

and their division and differentiation into corneocytes provide the bulk of skin’s

barrier function capacity. They actively divide in the stratum basale and mi-

grate through the upper layers while terminally differentiating. The outwards

cellular movement pushes any substances that may have breeched the stratum


corneum out to the surface, which may include NPs. However, no studies have

validated this possibility. As keratinocytes move outwards and prepare to be part

of the stratum corneum, they reach a stage known as the stratum granulosum

where they produce keratinohyalin and lamellar granules that contain character-

istic stratum corneum substances like keratin, filaggrin, loricrin, and lipids. These

proteins give the stratum corneum its anionic nature while ensuring proper me-

chanical properties. The intercellular lipid lamellae are formed to provide water

resistance in the stratum corneum, and consist mostly of ceramide, fatty acids,

and cholesterol along with the natural moisturizing factors that keep the stra-

tum corneum hydrated. [57] One of the most important changes is the formation of

tight intercellular junctions using desmosomes to link intermediate filaments and

E-cadherin junctions to link actin filaments. This connection between neighbor-

ing cells provides physical strength and unity of the stratum corneum barrier. As

keratinocytes integrate into the stratum corneum, they lose their nuclei, flatten

into corneocytes, and undergo a crosslinking step that binds all the interior and

extracellular proteins to the membrane, forming the cornified envelope. The final

result is a brick and mortar structure (Figure 1.2) consisting of 12-16 layers of

corneocytes with intercellular lipids.

Once fully mature, the stratum corneum provides an effective barrier with

its dense cellular cornified envelope, tight corneodesmosome junctions, and most

importantly its intercellular lipid lamellae. The glands and stratum corneum

proteins provide other methods of resisting permeation through an acidic surface

pH (∼5.0) and an ionic nature that forces most potential penetrants into an

unstable state. [27,58] For substances to gain entry into the epidermal cellular layer

and the body system, they have to cross this barrier. Toxicological concerns and

applications in transdermal delivery have encouraged the study and modelling of


Figure 1.2: Skin ArchitectureSchematic illustrating the multilayer architecture of skin adapted from Bouwstraet al.[16] The layers of the stratum corneum provide a barrier to insult. However,both hydrophobic and hydrophilic pathways exist for small molecules. The layersof the epidermis basal to the stratum corneum provide an apical developmentalflow of cells that adds to the barrier function.


skin penetration for a large variety of molecular substances. [59,60] Experimental

and modelling results have shown that the penetration of substances is driven by

surface energy (i.e. hydrophobicity/hydrophilicity) and size. These factors allow

penetration through the stratum corneum barrier via several pathways, but the

low levels of penetration in healthy skin have not allowed the extension to NP

skin penetration. However, some modeling efforts have been made to understand

the effect of sonophoresis/sodium lauryl sulfate treatment and suggested that

barrier abrogation induces pore opening through the stratum corneum that allows

increased NP permeation. [33]

1.3.2 Acute Injury vs. UVB Barrier Disruption Mecha-

nisms

Most skin barrier abrogation techniques studied in relationship to QD skin pen-

etration work in an immediate fashion by removing stratum corneum lipids or

opening holes in the stratum corneum and stratum granulosum layers of the epi-

dermis. Impairment methods such as tape stripping and dermal abrasion have

a peak effect soon after engagement, and the subsequent repair response begins

immediately thereafter in a well characterized fashion. [61,62] The initial component

of this is a rapid release of lamellar granules in the late stratum corneum to pro-

vide as much barrier replenishing lipid as possible. [63] To help fill the remaining

defects, mRNA and enzyme activity is subsequently increased by calcium gradi-

ent loss [64–66] and interleukin production (specifically IL-1 and IL-6). [67,68] These

activities stimulate synthesis of DNA to replenish damaged cells and synthesis

of the major stratum corneum components: fatty acids, ceramides, and choles-

terol. [61,66,69–71]


However, when skin is exposed to an acute dose of UVB the barrier impairment

follows a different path. Immediately after exposure there is no discernible bar-

rier damage, but a flood of signaling molecules is released that strongly impacts

the basal proliferative keratinocytes, including cytokines and prostaglandins. [72,73]

Additionally, there is internalization of the basal cell epidermal growth factor re-

ceptor (EGFR) and downstream activation of keratinocyte growth factor receptor

(KGFR) in the suprabasal differentiated keratinocytes, mechanisms that increase

phagocytosis of melanosomes from neighboring keratinocytes in response to UV

exposure. [74,75] Soon thereafter, a strong increase in DNA synthesis occurs as the

basal keratinocytes begin to rapidly proliferate in a similar fashion to other acute

injuries. [76–79] Corresponding to this increase is a prostaglandin-driven decrease

in the E-cadherins junctions, which allows the newly proliferated cells to migrate

more quickly to the stratum corneum surface. [80] Barrier disruption as measured

by TEWL has a long lag time, and occurs after DNA synthesis, a significant

difference between UV and other skin barrier disruptions. The barrier disrup-

tion process begins at about three days after UVB exposure and reaches a peak

within the next 1-2 days [78,81,82] (Figure 1.3) with a similar magnitude as the treat-

ment of skin with common barrier disrupting agents (i.e. acetone, tape stripping,

and surfactant treatments). [83,84] The mechanism of UV barrier abrogation is not

completely clear, but has been suggested to be due to a immune cell mediated

pathway [78] and is correlated to a loss of the epidermal calcium gradient (also

important for the cadherins junctions), [85] a disorganization of stratum corneum

lipids, [82] and the appearance of inadequately differentiated cells in the stratum

corneum (i.e. the appearance of nuclei). [86] After 7-8 days, the last of the damaged

keratinocytes are sloughed off and skin barrier status returns to normal. The pos-

sibility of exposure to UVB and NPs over the time period which has a substantial


barrier defect suggests the need to understand this phenomenon in vivo. These

profound effects of UVB motivate investigation of its impact on skin penetration

and QD/cell interaction, which is the focus of this doctoral dissertation.

1.4 The Nano-Bio Interaction

1.4.1 NP Skin Penetration

The skin has had a great amount of research effort focused on determining the

ability of NPs to penetrate intact and damaged barrier. The most well studied

group of NPs is that of TiO2 and ZnO, which are commonly used in cosmetics

such as ultraviolet radiation (UVR) protective sunscreens. Since UVR has long

been known to have a barrier impairing effect, [17] frequent use of sunscreens is

likely to occur on sun-damaged skin where penetration is more likely. Moreover,

there is little standardization in the NP physicochemical properties (size, surface

characteristics etc) or vehicle used in sunscreen formulation. These characteristics

may greatly impact NP skin penetration risk. The earliest studies on acute NP

skin interaction were of micro-fine titanium dioxide whose size was not specified,

and evidence of significant penetration was not found after repeated exposure to

human subjects. [22] Most subsequent research using acute exposure of NP-sized

TiO2 with sizes ranging from 10-100 nm diameter has supported the findings of

minimal penetration past the stratum corneum for NPs delivered in oil/water

emulsion for hydrophilically coated, [20,21] hydrophobically coated, [19,21,87,88] and

uncoated NPs, [89] mostly using human skin. ZnO NPs with a size range of 20-100

nm have been applied similarly in oil/water emulsions, and minimal penetration

observed for hydrophilically coated, [23] hydrophobically coated, [90] and uncoated


NPs [20,24] in human skin. A variety of other metal NPs have also been examined

with similar results. Maghemite and iron NPs with a particle sizes of 5 nm and

50 nm were determined to minimally penetrate ex vivo human skin in a static dif-

fusion cell. [27,91] For QD, research on ex vivo porcine skin using neutrally charged

PEG-ylated nail-shaped QD with a 40 nm hydrodynamic diameter; [13] ex vivo rat

skin using negatively charged carboxylic acid coated round and ellipsoidal QD with

14 nm and 18 nm hydrodynamic diameter, respectively; [29] ex vivo human skin

using carboxylic acid coated round QD with 14 nm hydrodynamic diameter; [31] ex

vivo human skin and in vivo SKH-1 hairless mouse skin using PEG-ylated nail-

shaped QD with a 40 nm hydrodynamic diameter [15] has supported an inability

of QD to penetrate the intact skin barrier. The NPs mentioned thus far are all

hard, insoluble and have metallic cores, and can be considered distinct from the

soft NPs normally made from carbon-based polymer components. This discussion

of skin penetration will be limited in scope to the mechanically and chemically

similar hard, insoluble metallic NPs.

Despite the large number of studies demonstrating a lack of NP skin pene-

tration, some studies have found that acute treatment of healthy skin with NPs

can allow penetration. For example, the first study to examine the skin penetra-

tion of QDs in 2006 was by the Monteiro-Riviere research group employing an ex

vivo porcine skin model. [42] Their initial results found that within 8 hours to 24

hours of QD exposure, ex vivo porcine skin had a large amount of QD penetration

throughout the epidermis and deep into the dermis in some cases for positively,

negatively, and neutrally charged QDs with round and ellipsoid shapes and hy-

drodynamic diameters of 14-45 nm in basic borate buffer. These results contrast

with most of the subsequent QD literature quoted in the above paragraph, as well

as more recent follow-up studies from the same group demonstrating minimal


QD skin penetration with the same QDs and application buffer with ex vivo rat

skin [29] and nail-shaped PEG-ylated QDs in neutral pH buffer on ex vivo porcine

skin. [13] This evidence suggests that their early results may have been due to the

interaction of their strongly basic borate buffer application solution (pH=8.7-9.0)

with the stratum corneum barrier preferentially in porcine skin or quality of the

porcine skin used, particularly considering the extreme extent of observed QD per-

meation. Additionally, gold NPs have been suggested to penetrate healthy skin

when in a small enough size range. Sonavane et al. determined that in an ex vivo

rat skin model 15 nm hydrodynamic diameter gold NPs were able to penetrate

healthy skin efficiently. Penetration levels decreased in 102 nm gold NPs and were

almost eliminated with 198 nm gold NPs. [92] Another study has demonstrated the

ability of 5 nm gold NPs to effectively penetrate healthy skin, and were able to

use this characteristic to increase the co-penetration of FITC dye or proteins such

as HRP protein (45 kDa) and β-gal (650 kDa). [93] Huang’s work suggests a use

of this technique to enable vaccine development and postulates that the size of

their NPs is smaller than the 0.5-10 nm hydrophilic pores present in the stra-

tum corneum intercellular lipid lamellae. Since these two studies both support

the ability of gold NPs to penetrate healthy skin but do not address what might

induce the difference between their gold NPs and other types of hard, metallic,

insoluble NPs the subject warrants future research.

Despite these few examples otherwise, there is a consensus that most hard

metallic NPs are unable to appreciably penetrate healthy skin, but with barrier

disruption levels of skin penetration have the possibility of being increased. With

ZnO NPs, work by Kuo et al. has used second harmonic generation multiphoton

microscopy and whole tissue imaging to find low levels of NP skin penetration

with uncoated ZnO of 10 nm hydrodynamic diameter that is increased by skin


treatment with chemical penetration enhancers including oleic acid, ethanol, and

a mixture of both. [30] An advantage of second harmonic generation microscopy is

that signal is dependent on the particulate form of the analyte. Another barrier

disruption mechanism has been suggested to be repeated exposure to NPs. A

recent study by Wu et al. has examined in vivo porcine and murine skin with

uncoated TiO2 NPs ranging in size from 4 nm to 90 nm in anatase and rutile forms

and discovered that the daily application of titanium dioxide for 30-60 days allowed

penetration throughout the epidermis and collection in distal organs including the

liver and the brain, with the smallest 4 nm NPs demonstrating the highest lev-

els of penetration. [89] In addition to the organ collection trends, titanium dioxide

in the liver and skin induced oxidative stress as determined by increased lipid

peroxidation levels and reduced collagen content, which developed into patholog-

ical lesions. This is the first recent study examining repeated exposure effects of

nano-TiO2, and such drastic results clearly warrant future investigation; particu-

larly in light of a recent report that showed TiO2 NPs induce DNA damage and

genetic instabilities in an in vivo mouse model when ingested. [94] Another study

used isotopic 68Zn to synthesize ZnO NPs and microparticles that were repeatedly

applied to humans each day for 6 days. [95] After the second day (4 applications),

68Zn was found in the blood and urine samples of ZnO NP sunscreen receiving

volunteers through the end of the study, with a maximum of 0.71% of the ap-

plied dose being detectable in females. However, they are unable to determine

whether or not the NPs applied stay in particulate form or if the detected inter-

nalized zinc is from leached ions, which have been shown previously to penetrate

the skin when delivered in zinc salt form. [96] In keeping with low levels of skin

penetration, the earliest studies on acute NP skin interaction were of micro-fine

TiO2 whose size was not specified, and evidence of significant penetration was not


found after repeated exposure to human subjects. [22] Another recent study has

investigated the skin penetration of TiO2 NPs applied every day for one month

using mass spectroscopy on the liver, lymph nodes, epidermis, and dermis to find

that there were was minimal detectable skin penetration with a variety of NP

formulations. [97] The varied results reported highlight the possibility for an effect

of composition on skin penetration or a detection dependence on NP stability in

the acidic superficial stratum corneum environment.

The impact of skin barrier disruption has also been examined for QDs. An

early study in 2006 used low frequency sonophoresis to impair the skin barrier, a

common drug delivery enhancement technique, and found a substantial increase

in QD skin penetration (coating and hydrodynamic size not stated) in an ex vivo

porcine skin model. [32] Recent work has used a synergistic combination of sodium

lauryl sulfate and sonophoresis to increase the penetration of QDs with positive,

negative, and neutral charges and hydrodynamic diameters ranging from 10-20 nm

as well as 4.3 nm negatively charged gold NPs in full thickness and dermatomed ex

vivo porcine skin. [33,34] With QDs and no barrier disruption, their data suggested

low but quantifiable levels of penetration into the dermis using a physical technique

to separate the dermis and epidermis and quantitative QD evaluation by mass

spectroscopy. Research in our lab has found low levels of negatively charged QD

skin penetration with hydrodynamic diameter of 14 nm through healthy ex vivo

human skin with an increase in stratum corneum penetration when ex vivo human

skin is treated with a depilatory agent or tape stripped using flow cytometry. [31]

Interestingly, independently conducted research using nail-shaped PEG-ylated QD

with hydrodynamic diameter of 40 nm by Gopee et al. on in vivo mice [15] and

Zhang et al. on ex vivo rat skin [29] has found that tape stripping, a well-accepted

method of barrier disruption [98] and permeability enhancement of large hydrophilic


molecules, [84] had no impact on QD skin penetration. The difference in results

could indicate a possible advantage in sensitivity of flow cytometry techniques

over organ analysis [15] and limitations of histological analysis using fluorescence

confocal microscopy. [29] When the more aggressive barrier disrupting technique

of dermal abrasion was used, both groups were able to observe a substantial

increase in the skin penetration of QD. Dermal abrasion is an invasive technique

that eliminates the stratum corneum and much of the epidermis completely to

allow free access of QD to the dermis and vasculature. Other types of NPs, such

as anti-microbial nano-silver, have demonstrated skin penetration in vivo when

applied to a 30% burn victim using the Acticoat wound dressing with 25 nm

silver NPs. The patient’s daily exposure to nano-silver on strongly weakened skin

barrier yielded high serum levels of silver and caused a grey discoloration of the

skin. [99] In an ex vivo study, 25 nm silver NPs in a synthetic sweat formulation

with 10% ethanol were able to penetrate healthy skin in very low amounts, with

a 5-fold increase in ex vivo human skin penetration after dermal abrasion. [100]

Despite some differences in the detailed levels of impairment that are necessary

to effect the skin permeability to QD or other metallic NP that may be due to

particle size/shape, particle coating, or skin type, it is clear that disrupting the

skin barrier can increase risk of NP penetration.

1.4.2 QD Interaction With the Local Epidermal Cell Types

The studies discussed above suggest that depending on the NP characteristic and

the severity of the insults, barrier compromised skin can be breached by NPs.

After passing the stratum corneum barrier, NPs first encounter the local cel-

lular environment of the epidermis. The interaction of NPs with these cells is


of additional concern, as there is an expansive body of literature regarding the

toxicity of NPs. [37–40] These studies have clearly established non-specific cell up-

take, [101] receptor mediated cell uptake, [102] and cytotoxicity [103,104] of NPs (metal

oxides, quantum dots, carbon nanotubes, etc.) by many cell types including nerve

cells, [105] macrophage cells, [106] dermal fibroblasts, [107] keratinocytes [108] and oth-

ers. [105,109–111] Due to the large number of studies available in the literature for all

of the major nanomaterials, this section will be predominately limited to the NP

focused on in this thesis, the quantum dot, and on the dominant epidermal skin

cell types.

With respect to the skin, researchers have examined the interaction between

QDs and the cells most common in the epidermis: dendritic antigen presenting

cells, melanocytes, and keratinocytes. Work by Vogt et al. found that CD1a+

Langerhans cells, an important antigen-presenting immune cell type in the skin

that are commonly found lining the hair follicles, endocytosed 40 nm polymeric

particles when applied to the surface of the skin. [55] Work in our lab has determined

PbSe PEG-ylated QD toxicity to metastatic melanoma cells, [112] and further stud-

ies currently in preparation for publication examine the viability and reactive

oxygen species generation effects of QDs on melanocytes, which demonstrate sim-

ilar levels of toxicity as keratinocytes despite lower cellular uptake. Others have

used QDs functionalized with RGD peptide to target melanoma cells. [113] There

is a clear lack of QD interaction studies on melanocytes, but due to their status

as secretory cells and relative scarcity in the skin (approximately 1 melanocyte:

36 keratinocytes), the effect of QDs on keratinocytes is of greater interest. In

keratinocytes, however, very few studies exist. Ryman-Rasmussen et al. have

worked with human epidermal keratinocytes (HEKs), a proliferative-type ker-

atinocyte line, to determine the toxicity and uptake mechanisms for a variety of


commercially available QDs. [13,108,114,115] Their earlier 2007 in vitro studies found

non-specific cellular uptake that was independent of surface chemistry, a surface

chemistry dependent inflammatory cytokine release, and dose dependent cytotoxi-

city. [108,114] They suggest toxicity limits consistent with the literature for other cell

types for 565 nm round and 655 ellipsoidal QDs with positive, negative, and neu-

tral surface charges (between 2 nM and 20 nM at 24 hrs and 48 hrs for all surface

chemistries). Additionally, their recent work has suggested a putative keratinocyte

uptake mechanism for negatively charged QDs that is governed predominately by

proliferative keratinocyte low density lipoprotein receptors and G-protein cou-

pled receptors. [115] However, they did not see any effect of clathrin inhibition,

the commonly associated internalization mechanism for low density lipoprotein

receptors. [116,117] More work is required in this field to understand the uptake

mechanisms and the several compensatory pathways that may be involved. [118]

These results are important because if basal keratinocytes endocytose QDs, there

is an increased risk of cell death, dysfunction, or transformation, as these cells are

anchored to the basement membrane and produce daughter cells for long periods

of time in the epidermis. [119]

Due to their proximity to the surface of the body it is important to consider

the effects of environmental insults on epidermal cell interaction with NPs , such

as could occur with a combination of UVB exposure and a NP containing cos-

metic. However, very few of such studies exist in the literature. One notable

example is a recent study by Chang et al. that examined the impact of UVA on

pancreatic carcinoma cells that were pre-exposed to thiol-capped CdTe QD. [120]

They were able to find a substantial increase in cytotoxicity when cells were in-

cubated with QDs and then exposed to UVA. This data is interesting, but the

cell type, the pre-exposure with QD at high molarities, the extremely large doses


and wavelength (365 nm) of UVA used in the study (on the order of total UVA

dosage from 11.4 J/cm2 to 68.4 J/cm2), and the time frame of QD exposure (4

hours maximum) limit comparison with our study. Chang et al. did find that

with shorter pre-exposure to QDs there was much less impact on the QD toxicity,

which suggests that their cytotoxic effect may be due to internalized or degraded

QDs non-radiatively releasing energy by catalyzing reactive oxygen species gener-

ation. This finding is supported by previous work that found a strong increase in

the cytotoxicity of QDs that were exposed to high levels of UV light before incu-

bation with cells. [121] When other NP types are considered, several studies have

demonstrated a dramatic increase in ROS production by UVA light from both

the rutile and anatase crystal forms of TiO2, [111,122–125] however, the anatase form

is significantly cytotoxic even in the absence of UVA. [111,126] This is a concern as

the anatase form is common in the formulation of sunscreens. [127] Despite toxicity

issues, researchers have demonstrated QD tracking in live cells for over a week. [128]

However, in highly proliferative cells such as basal keratinocytes non-apoptotic re-

active species stress may be of greater concern than cytotoxic events, as reactive

oxygen species are well known to damage DNA and accumulation of these events

could allow for genetic transformation. [94,122,129,130]

1.4.3 Systemic Impact

If QDs are able to penetrate the skin, in addition to their local cellular inter-

action they will be distributed systemically. In addition to accidental exposure,

QDs and other NPs have been investigated for use in cancer diagnosis and treat-

ment in vivo. [3,131? ,132] Controlling NP systemic distribution to decrease toxicity,

commonly with the goal of renal clearance, is viewed as a key parameter in the


establishment of NPs for usage in the diagnosis and treatment of disease. The

majority of the literature on the subject examines the selective targeting of NP

treatment agents to tumors by means of passive or active targeting. [133] Since this

doctoral thesis deals with the the biological interaction of untargeted QDs, the

distribution thereof is of primary importance. Research by Frangioni’s group has

suggested that QDs with hydrodynamic diameter less than 5.5 nm in serum are

able to be efficiently excreted in the urine for untargeted and targeted QDs, using

zwitterionic [45,131] and PEG [3] coatings. Since the QDs and other NPs used in

research and consumer products have a larger size than that, their biodistribu-

tion profiles tend to change. In an early study Akerman et al. found that tail

vein injected QDs functionalized with mercaptoacetic acid and targeting peptides

demonstrated collection levels in the liver and spleen that could be reduced with

the inclusion of PEG ligands on the surface of CdSe/ZnS QDs. [102] Subsequent

work by Ballou et al. found that PEG coated CdSe/ZnS QDs injected by tail

vein migrate to the liver, spleen, bone marrow, and lymph nodes and can be

detected as much as 2 years later. [132,134] Work by Fischer et al. found that mer-

captoundecanoic and BSA coated CdSe/ZnS QDs were distributed mostly to the

liver, with some presence in the spleen and lung that was dependent on surface

coating at a time point of 90 min post-injection. [47] The presence of PEG-ylated

QDs injected by tail vein was further examined by Yang et al., who found col-

lection in the liver and spleen up to 28 days after injection. [135] Another study

examined PEG-ylated and carboxylated QDs that were tagged with 64Cu to allow

imaging using microPET, and found uptake in the liver and spleen with both QD

functionalizations and additional bone marrow uptake with the PEG-ylated QDs

within a few minutes of tail vein injection. [136] A recent study on the collection

of QDs after tail vein injection found strong collection of PEG-ylated rod shaped


CdSe/ZnS QDs in the liver that faded within 2-5 days, bone marrow that faded

within 3-6 months, and lymph nodes that remained for two years with presence

of QD fluorescence able to be detected in the liver, spleen, and lymph nodes with

spectral blue shifting after the two year time span. [137] However, the applicability

of these studies to the fate of QDs that diffuse into the dermis and are distributed

systemically is limited, and more directly related subcutaneous injection studies

are needed to predict the systemic fate of skin penetrating QDs. Gopee et al.

have shown that subcutaneous injection of PEG-ylated nail shaped CdSe/CdS

QDs with hydrodynamic diameter of 40 nm collect in the liver and lymph nodes

consistently at 24 and 48 hours using mass spectrometry to detect Cd. [36] Their

study found additional collection of QDs in the kidneys and spleen at 24 hours,

but the relative amount was lower (i.e. percent of applied dose and ng Cd/ g tis-

sue). Their subsequent work investigating the skin penetration of the same QDs

found the Cd lymph and liver collection at 24 hours to be optimal for evaluation

of the skin penetration, and was able to detect the penetration of QDs through

dermally abraded skin. [15] The consistency and increased gram Cd per gram tissue

measurements found in the liver and lymph nodes at 24 hours after QD exposure

provide guidance for the techniques chosen to evaluate QD skin penetration in

barrier disrupted skin discussed in this thesis.

1.5 Evaluation of Skin Barrier Function

A primary challenge when attempting to understand the ability of barrier im-

pairment to affect skin penetration of NPs is in quantifying the degree of barrier

impairment. It is of particular importance when attempting to predict the impact

of a barrier disruption treatment or standardizing the amount of damage that is


induced with different types of barrier disrupting agents. There are two main tech-

niques to evaluate skin barrier status- skin resistivity and transepidermal water

loss (TEWL). Skin resistivity is commonly used to determine the viability of ex

vivo skin samples, and generally consists of two electrodes, one on the apical and

one on the basal sides of the skin. An ex vivo skin sample is then either pulsed with

an AC voltage or current at standard frequencies and the resistance measured, or

the voltage is clamped and the equilibrium resistance determined. The resistances

commonly defined for healthy skin in various species are well accepted, [138] and

the technique is used in a wide variety of NP penetration literature [27,31,33,34] as

well as the molecular skin penetration literature. [59,139–142]

However, when in vivo barrier measurements are considered, TEWL is the

preferred measurement technique. TEWL measurement is a well established tech-

nique that functions by measuring the flux of water vapor out of the skin over a

controlled area. A number of studies have detailed the correlation between TEWL

measurements and permeability of skin compromised by chemical means (such as

sodium lauryl sulfate [143] or acetone [83]), by physical means (such as tape strip-

ping or skin surface biopsy [144]), or by means of UVR exposure. [78,82,85,86] Some

researchers have gone further, and have correlated TEWL measurements with

the permeability of skin to small molecules based on hydrophilicity. [83,145,146] No

studies, however, have examined the relationship between defective skin barrier

function as measured by TEWL and skin permeability to NP. Previous work us-

ing increasing molecular weight PEG molecules (300 Da, 600 Da, and 1000 Da)

suggests that with a given barrier disruption such as tape stripping, TEWL can

predict the permeation behavior of even the largest PEG molecules, [84] but this

has not been extended to the range of NPs. UVB exposure has been demonstrated

to increase TEWL to similar levels as are observed with chemical and physical


barrier disruption (Figure 1.3), [78,82,85,86,147] and some very early literature has

suggested that this can impact the skin permeability to hydrocortisone, [147] but

the relationship between UVB and skin permeability has not recently been ex-

plored despite the importance in exposure to sunscreen agents. Since there are no

studies in the literature that address the effect of UVB exposure on NP skin per-

meability, the research presented in this doctoral dissertation on the penetration

of QDs through UVB compromised skin over the relevant time period will fill an

important void.

1.6 Evaluation of In Vivo QD Skin Penetration

The delayed barrier impairment response induced by UVB exposure necessitates

its in vivo investigation, and thereby eliminates the possibility for usage of com-

mon quantification techniques that have been implemented in ex vivo NP skin

penetration studies. Additionally, the low penetration levels commonly observed

in NP skin penetration studies make traditional evaluation techniques challenging

to implement, and results in the quality of the current literature available on skin

penetration of NPs (e.g. QDs) being limited in a large part by the analytic tech-

niques employed for analysis. For this reason, there is a constant effort to decrease

noise factors by improving sensitivity, eliminate background, and increase signal to

noise ratio. Current state of the art techniques include histological processing and

imaging; electron microscopies; mass spectrometry; and advanced microscopies

such as multi-photon or confocal microscopy.


TEW

L (g

/m2 /h

)

40

30

20

10

00 1 2 3 4 5 6 7 8 9 10Days after UVB Irradiation

*** ** *

**

Figure 1.3: UVB Induced TEWL DefectTEWL curve adapted from Jiang et al. detailing response after exposure of SKH-1to 150 mJ/cm2 UVB.[136] *P<0.05 and **P<0.01. Note the peak values ∼4 daysafter UVR exposure. Values reported are Mean±S.E.M. for n=6.


1.6.1 Histological Sectioning

The traditional standard for evaluation of in vivo skin penetration, and most other

dermatological studies, is that of histology. Histological techniques have been in

active use since at least the publication of the first histological textbook in 1852

by Rudolph Von Koelliker, Handbuch der Gebewelehre. In this technique, the tis-

sue of interest is frozen or fixed and embedded in optimum cutting temperature

(O.C.T.) compound or paraffin that allows for cutting of tissue sections at 5-20 µm

thickness, and subsequent mounting and staining procedures as needed. For the

evaluation of NP penetration, cryo-sectioning is most commonly used to decrease

the amount of processing necessary that might diminish QD signal. To analyze

skin penetration of QDs or other fluorescent NPs in histological sections, widefield

fluorescence microscopy and confocal microscopy are normally implemented in a

very similar fashion. Researchers commonly use confocal microscopy with histol-

ogy to eliminate possibility of slicing contamination of samples by imaging only the

center of a histological cryosection. [13,15] Similar performance on sectioned tissue

is easily achievable if care is taken with slicing direction and blade replacement, as

will be performed in this doctoral thesis. [14] In both scenarios, background noise

and QD brightness is adjusted by laser wavelength or lamp filter choice and out-

put bandpass filter. Even when imaging in the red visible wavelengths, there are

substantial challenges with tissue autofluorescence that limit the ability of fluo-

rescent NPs such as QD to be conclusively separated from the surrounding tissue.

For example, a fluorescent NP on the edge of a layer of the cornified envelope

can clearly be seen to be brighter than the surrounding area (Figure 1.4A). When

a line profile of the area is analyzed, the separation in intensity becomes clear,

and an appropriate image threshold can be determined (Figure 1.4B). However,


the resultant image is unable to be analyzed for QD skin permeation as the sig-

nal outside of the area immediately surrounding the putative QD is drowned out

by autofluorescence (Figure 1.4C). This problem is common to all visible range

fluorescent imaging, whether widefield or confocal, but the acceptance of the his-

tological technique and the ability to visualize the fluorescence of QD in the tissue

with a minimum of microscopy knowledge offers advantages that ensure its use

for the foreseeable future. To limit this issue, one thrust of this thesis proposes a

near-IR confocal microscope that will use a near-IR quantum dot and excitation

source, which has the additional benefit of advantageous whole tissue imaging,

technical background of which will be described in Section 1.6.4.

1.6.2 Transmission Electron Microscopy

Transmission electron microscopy (TEM) is an electron microscopy that functions

by focusing a powerful beam of electrons on a sample in a vacuum. The sample

then absorbs a portion of the electrons and the resultant information can be

collected by a camera. TEM was developed in the 1970s, and are able to provide

important ultra-structural analysis of NP skin penetration. TEM allows for very

detailed analysis of sub-cellular NP localization, and can enable the imaging of

single NPs as well as elemental analysis to confirm the presence of the element

of interest. The primary limitations of the TEM technique are time, cost of

processing, sample size, and variability of enhancement procedures. TEM slices

tissue to a thickness of ∼70-100 nm, so it is difficult to achieve a large sampling

area. For example, to sample only ∼1 mm2 of skin would require analysis of on

the order of 1000 slices. The protocol used in this thesis requires >30 different

fixing, washing, and staining steps before the sample is embedded in resin, so


50

60

70

80

90

100

110

120

0 10 20 30 40 50

Abrit

rary

Flu

ores

cenc

e Un

its

Line Profile

C

B

A

Figure 1.4: Visible-Range Tissue AutofluorescenceFluorescent imaging in the visible regime presents challenges due to tissue autoflu-orescence. A single 625 nm emitting quantum dot (or small clump) can be imaged(A) and in the blowup be seen to be clearly distinct from its surrounding area.The line profile (B) provides proof and allows for image thresholding (red dottedline). However, the thresholded image (C) shows that there are many areas wherequantum dots of a similar fluorescent level could be washed out with backgroundautofluorescence. Scale bars in (A) bottom left and (C) bottom right are 25 µm.


there is an increased opportunity for data loss and error as compared to the 2-3

fixing and staining steps used in cryo-histology. Despite its considerable power

in evaluation of ultra-structural location of skin penetration, TEM’s limitation to

analysis of very small tissue sections makes quantification difficult and results are

commonly reported as supporting data as will be done in this thesis. [14,29,32]

1.6.3 Elemental Analysis

Atomic absorption spectroscopy and mass spectroscopy have been widely used for

a number of years, and have a number of advantages including very good sensi-

tivity and quantification of absolute NP presence. However, they are destructive

techniques and so can only be used on whole organs or skin samples that are

separated into transverse layers (usually by tape stripping at completion of the

experiment) to determine depth penetration of NPs. Early work showing mini-

mal skin penetration of nano or micro-TiO2 was performed using the tape strip

analysis technique, [19] but the reliability of a positive result would be questionable

due to the known collection of NPs along the hair shaft and in the hair follicle,

which extends into the dermis. [26,55] As such, the analysis is more suited to the

study of systemic distribution in vivo. A recent study showed minimal penetration

of PEG-coated CdSe quantum dots through intact mouse skin with a notable in-

crease after barrier disruption (tape stripping and dermal abrasion) using analysis

of the lymph nodes and liver for Cd, [15] and another showed minimal penetration

through the skin for TiO2 with repeated application to pigs in vivo. [97] To harvest

the distal organs, the animals are carefully dissected using sterile technique and

nitric acid treatment of the dissection instruments to prevent contamination of

samples, with dissection moving from control animals to those that have been


treated with the analyte of interest. The liver (Figure 1.5A) is a large organ that

can be removed easily, but care must be taken to to ensure complete separation

without destruction of the brachial lymph nodes (Figure 1.5B) and axillary lymph

nodes (Figure 1.5C). The primary limitation of the technique is that only a small

percentage of the applied dose is trafficked to the distal organs. For example, an

earlier study by Gopee et al. found that only 6% of a subcutaneously injected

dose was able to be transported to the liver and 1% to the regional lymph nodes

at 24 hours. [36] Mass spectroscopy and atomic absorption spectroscopy have the

advantage of quantification, but do not allow localization of NPs in cellular skin

structures (hair follicles, cell types, etc.) or affirmative determination of skin layer

(stratum corneum, terminally differentiating epidermis, basal epidermis, or der-

mis). Thus, atomic absorption will be used in this thesis to determine the system

level penetration of QDs through UVB exposed skin.

1.6.4 Advanced Microscopy

All of the techniques discussed thus far require extensive tissue processing and

have strong limitations in the ability to sample an adequate area, provide cellular

level resolution, or differentiate dissolved ions from intact NPs. When attempting

to evaluate skin penetration in a toxicological context low frequency events may

be important and so factors such as limit of detection and the state of the NP (i.e.

dissolved ions or intact NPs) in the skin become increasingly critical. One of the

most promising ways to address these problems are through imaging modalities.

For imaging of NPs, the available modalities depend on the physical characteris-

tics of the NP. The most commonly implemented form of advanced microscopy

is that of fluorescent confocal microscopy. Fluorescent confocal imaging provides


A B

C

Figure 1.5: Distal Organ LocationThe liver and lymph nodes in the mouse can be efficiently removed with carefuldissection technique. The liver (A) is a large organ, and all lobes are collectedfor elemental analysis. The brachial (B) and axillary (C) lymph nodes are theregional draining lymph nodes for topically applied substances on the back, andwith care can be found and removed consistently.


advantages in the exclusion of defocused light using a pinhole, and therefore allows

optical sectioning of samples. Despite its ubiquity confocal microscopy is often

is relegated to analysis of histological samples in practice, but some examples of

whole tissue microscopy have been reported. Richard Guy’s group has been one of

the pioneers in this area, using confocal microscopy to evaluate the skin penetra-

tion of a variety of fluorescent polymeric NPs. [26,148–150] For example, a 2004 study

by Alvarez-Roman et al. found that FITC labeled polystyrene NPs of 20 nm and

200 nm diameter were unable to penetrate porcine stratum corneum. [26,148] For

QDs, whole tissue confocal microscopy has been used by Robert Langer’s group to

support reported increased skin penetration of QD with sonophoresis and sodium

lauryl sulfate treatment. [34] However, in all of these studies, visible range whole

tissue confocal microscopy has been relegated to a minor supporting technique as

epidermal scattering and absorption limit the penetration depth of light, both in

the acceptable laser excitation powers as well as the returned fluorescence. Mul-

tiphoton microscopy overcomes the excitation and average power limitations of

visible range confocal microscopy by using femtosecond pulses of near-IR light at

twice the excitation wavelength, thereby achieving greater imaging depth in the

skin, but at the expense of resolution. The technique functions by the genera-

tion of phenomena such as 2-photon fluorescent excitation or second harmonic

generation of the target analyte, and has been demonstrated to detect ZnO NPs

using second harmonic generation. [24,90,151] One study has even been able to put

the technique into practice and demonstrate an increased skin penetration of ZnO

NPs with barrier disruption by chemical penetration enhancers. [30] These studies

are important, as ZnO is an important particle that is commonly used in topically

applied sunscreens, but its dominant emission peak at 385 nm limits the detec-

tion depth achievable. To address these issues, other techniques such as optical


coherence tomography and coherent anti-stokes raman spectroscopy have been

implemented, but little literature exists on their application in the skin. [152–154]

In addition to its examination of QD skin penetration with well established

models, this thesis proposes an all near-IR whole tissue reflectance and fluores-

cence confocal microscopy system with PbS based near-IR emitting QDs and

a near-IR excitation source. Confocal microscopy is a well established technique

that scans a laser across the field of view using a two dimensional mirror array and

collects the reflectance or fluorescence signal from the sample using an avalanche

photodiode or a photomultiplier tube with a pinhole to remove defocused light.

The presence of a pinhole allows axial slicing and localization of structures within

the tissue. The inclusion of a reflectance mode provides further advantages, be-

cause previous NP evaluation studies have used tissue autofluorescence to localize

NP penetration, which is unable to determine cell borders and differentiate the

layers of the skin. Reflectance confocal microscopy provides sub-cellular resolu-

tion and superior depth penetration without the need for standard tissue staining,

and is in active clinical use. [155–159] Movement into the near-IR tissue window, a

wavelength span (∼650-900nm) where there are no major tissue autofluorescence,

absorption, or scattering components increases the penetration depth and signal

to noise ratio for a whole tissue microscopy system. [160,161] The use of whole tis-

sue near-IR fluorescence and reflectance for the localization of QDs in this thesis

will expand the integration of technology in the skin penetration literature and

overcome the limitations of visible range whole tissue microscopies.


1.7 Statistical Evaluation

In this thesis, standard statistical analysis techniques were used including power

analysis to determine feasibility of experiments, full factorial designed experi-

ments with ANOVA analysis, and t-tests. Power analysis is a technique that this

dissertation used to determine the feasibility of experiments and determine the

necessary n-value for experiments. Power analysis is a technique that balances

the possibility of the two types of errors that can occur when statistically testing

a null hypothesis. Take for example the TEWL data depicted above (Figure 1.3).

If a null hypothesis is made that there is no difference in the measured TEWL

level at day 4 after exposure, then the alternate hypothesis would be that there

is a difference. A Type I error occurs if the average value at day 4 was found to

be different by statistical testing when in fact it was not, and is known as a false

positive. This value is commonly represented as α, and is the most commonly

evaluated error. A Type II error would occur if no difference was found at day

4 when there was in fact a physiologically relevant change, and is known as a

false negative. The Type II error is referred to as β, and 1 − β is the statistical

power of a test. To perform a power analysis, the probability of finding a Type I

and a Type II error are balanced. Power analysis is a quite useful technique that

allows a researcher to use literature or pilot study estimates of mean change in a

measured value and standard deviation to find the number of replicates needed

to achieve appropriate probabilities of making a Type I (normally α < 0.05) or a

Type II (normally 1−β > 0.80) error. For example, in the TEWL depicted above

(Figure 1.3), the peak mean value at day 4 is µ∗ = 32 g/m2/h with a standard

deviation of σ = 7.4, and the baseline value is µ0 = 11 g/m2/h. If we wished

to repeat this experiment and attain rigorous statistical values of α < 0.01 and


α Zα

0.001 -3.090.01 -2.330.05 -1.640.10 -1.28

β Zβ

0.01 -2.330.05 -1.640.10 -1.280.20 -0.84

Table 1.1: Common Zα and Zβ ValuesCommonly used α and β values with their corresponding Zα and Zβ values.

1− β > 0.95, the number of animals needed can be calculated using the following

power analysis formula.

n =

((|Zα|+ |Zβ|)σ|µ∗ − µ0|

)2

(1.1)

The variable Zα is defined as the x-value of the standard normal distribution

(which is a Gaussian curve centered at 0 with standard deviation equal to 1 and

integral equal to 1) that would yield an integral from −∞ → Zα equal to α.

The term is defined in the same fashion for Zβ, and these values are commonly

reported in standard normal distribution tables. [162] For ease of reference, some

commonly used α and β values with their corresponding Zα and Zβ are included

herein (Table 1.1). In the described example, this estimation provides a necessary

n of 1.96, so 2 animals would be needed to reached the desired confidence levels.

This low number is due to the large fold change in the mean of TEWL induced

by UVB exposure, and experimentally it is wise to include a margin of error due

to possibility of dropped replicates or above expected standard deviations. The

evaluation of statistical power is an important strategy for planning experiments

and ensuring that an expected experimental difference will be resolvable with a

practical number of experiments. [162]

With an accurate approximation of n for the experiment, the control factors

to be tested can be decided. If only one factor is to be evaluated, a standard


experimental design is adequate. For single-factor experiments, the use of t-tests

such as the Students paired and unpaired t-tests allow comparison of two Gaus-

sian distributions of data. However, if two or more factors are to be compared at

a time then ANOVA analysis and appropriate experimental planning is required

before execution. The most simple method is that of a full-factorial array, which

tests every combination of factors. A full-factorial array yields results on the sta-

tistical importance of each control factor and all interactions- another advantage

over traditional one at a time testing. Statistical results from a designed ex-

periment can be calculated manually, [163] or are more commonly evaluated using

software such as Matlab. If a large number of control factors are to be evaluated,

then more complex experimental guidelines are used to determine the trade-off

between certainty of the statistical model and the number of required treatment

conditions. [163] The designed experiments presented in this thesis all used full-

factorial arrays to ensure full evaluation of interactions. Readers interested in

learning more should consult the text Practical Guide to Designed Experiments:

A Unified Modular Approach, by Paul Funkenbusch.

1.8 Thesis Organization

The overarching goal of this thesis is to determine the effect of external stress

on NP interaction in the skin. In particular, UVB exposure is hypothesized to

increase the skin penetration of QDs. Herein are presented results of investigation

into the ability of UVB to impact the interaction of QD with skin barrier function

and local epidermal keratinocytes, and efforts towards increasing the state of the

art in terms of applied confocal fluorescence imaging in the skin.

In the first chapter of research entitledUVB and Immediate QD Skin Permeability-


Qualitative evaluation of UVB induced barrier defect on in vivo skin penetration

of QDs, the hypothesis that UVB increases skin penetration of QDs is evaluated

with 24 hour QD incubation immediately after irradiation. Results demonstrate

for the first time that UVB barrier disruption impacts skin permeability to NPs.

This thesis next endeavors to quantify the delayed impact of UVB on skin

barrier function and skin penetration of QDs in a chapter entitled UVB and De-

layed QD Skin Permeability- Quantification of QD skin penetration risk with UVB

induced delayed barrier impairment as measured by TEWL. Results of this study

demonstrate quantitative evaluation of UVB impact on inside-out barrier function

and outside-in penetration of QDs.

The previous sections of this thesis have supported the hypothesis that UVB

increases skin penetration of QDs, which may increase QD interaction with epider-

mal keratinocytes. In this chapter entitledUVB, QDs, and Primary Keratinocytes-

The impact of UVB and differentiation state on QD interactions with primary

keratinocytes, the effects of UVB and primary keratinocyte differentiation state

on QD cellular uptake were investigated to evaluate the hypothesis that UVB

increased QD association with proliferative and differentiated keratinocytes.

The final goal of this dissertation is to overcome challenges in the histological

evaluation of QD skin penetration through the development of a near-IR whole

tissue confocal microscope in a chapter entitled Near-IR QD Confocal Imaging-

The design, validation, and implementation of a combined reflectance and fluores-

cence mode near-IR confocal microscope for the imaging of QDs in mammalian

skin. This section has obtained promising results in the imaging of near-IR QD

skin penetration through human epidermis.

The final chapter of this dissertation summarizes the important findings of my

research. The important advancements presented in the thesis have contributed


novel findings to the skin penetration literature and increased awareness about

the impact of UVB on NP skin permeability, and opened new avenues of research

for future work investigating NP characteristic impact on skin penetration and

targeting to the epidermis.


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65

Chapter 2

UVB and Immediate QD Skin

Permeability

Qualitative evaluation of UVB induced bar-rier defect on in vivo skin penetration ofQDs 1

1Portions of this chapter are adapted from Mortensen, L.J., Oberdörster, G., Pentland, A.P.,and DeLouise, L.A. In vivo skin penetration of quantum dot nanoparticles in the murine model:the effect of UVB, Nano Lett 2008, 8, 2779.

CHAPTER 2. UVB AND IMMEDIATE QD SKIN PERMEABILITY 66

2.1 Introduction

As motivated in the introduction to this thesis, the skin penetration of nanopar-

ticles (NPs) is an important field that is growing due to NP use in applications

ranging from targeted fluorescent labels in the life sciences, [1] ultraviolet B ra-

diation (UVB) protective cosmetics, [2] and bacterial inhibitors in food storage

containers [3] to wound care products and baby pacifiers. Concurrent with this

growth, however, are increasing environmental and human health concerns. [4,5]

Of particular concern are UVB protective cosmetics and sunscreens. These con-

sumer products often contain significant amounts (∼5-10% by weight) of ZnO and

TiO2 NPs (<20 nm diam.) [6] and they are marketed to diverse consumer groups

(children and adults) for use on a daily basis. SPF (sun protection factor) con-

taining products are often applied on a repeated basis to skin that has suffered

sun exposure sufficient enough to have initiated biological UVB-induced skin re-

pair processes. Therefore, it is important to ascertain the potential effect of UVB

exposure on the skin barrier function with respect to NP penetration. Indeed,

as discussed in more detail in the introduction, a consensus is building in the

literature that intact skin is a sufficient barrier to prevent the skin penetration of

the majority of topically applied QDs [7–9] and other NPs; [10–13] but skin barrier

disruption can allow increased NP penetration. [7,8,14–16] The evaluation of UVB

impact on the permeation process is an important subject, as potential toxicolog-

ical consequences may result if NP are taken up by epidermal skin cells and/or

translocated to secondary sites.

In vitro studies have clearly established nonspecific cell uptake, [17] receptor me-

diated cell uptake, [1] and cytotoxicity [18,19] of nanoparticles (metal oxides, quan-

tum dots, carbon nanotubes, etc.) by many cell types including nerve cells, [20]


macrophage cells, [21] dermal fibroblasts, [22] keratinocytes, [23] and others. [20,24–26]

Early in vitro studies performed to assess the effects of QD on keratinocytes found

nonspecific cellular uptake that was independent of surface chemistry, a surface

chemistry dependent inflammatory cytokine release, and dose dependent cytotox-

icity. [23] It is a common finding that nanoparticle cytotoxicity results from the

generation of reactive oxygen species (ROS). [26–28] In the case of TiO2 however,

some studies fail to observe notable ROS generation. [27–29] This discrepancy likely

relates to the dependence of ROS generation on TiO2 crystal composition and to

differences in experimental UVA light levels. UVA light dramatically increases

ROS production from both the rutile and anatase crystal forms of TiO2; [26–28,30,31]

however, anatase is significantly cytotoxic even in the absence of UVA. [26,32] This

is a concern as the anatase form is common in the formulation of sunscreens. [2]

Given the ability of NPs to induce free radical cytotoxicity, the uncertainty re-

garding potential NP skin penetration within the ex vivo model and the limited

availability of in vivo data, especially in barrier compromised skin, examination

of an in vivo and UVB exposure model is necessary.

This chapter presents initial efforts to develop an in vivo model of NP skin pen-

etration employing SKH-1 hairless mice and quantum dot nanoparticles (QDs).

QDs have been selected for their ideal characteristics for in vivo experimenta-

tion including broad excitability, narrow emission bandwidth, high fluorescence

quantum yield, photostability, and ease of surface functionalization. [33,34] More-

over, QDs are of a similar size to TiO2 NP used in sunscreen applications, they

intrinsically generate ROS species, [18] and the carboxy terminated QDs have a

similar negative oxide surface chemistry to the TiO2 and ZnO raw materials of-

ten used in sunscreen applications. [13] QD interaction with skin is also of primary

interest, as the occurrence of QDs in the life sciences and other technical applica-


tions is rising [4] and has consequently increased the risk of QD skin exposure to

manufacturers, researchers, and consumers. We have selected the SKH-1 mouse

for these studies as it is a well-accepted model system for studying skin barrier

function, UVB induced skin cancer, and other diseases due to its similar follic-

ular density and dermal to epidermal differentiation. [35] In previous studies, the

biological effect of UVB irradiation has been examined at a variety of acute and

repeated doses. [36,37] Here, we are interested primarily in acute UVB exposure, as

it is the most applicable to consumer use and has been shown to have a signif-

icant effect on skin barrier function at a range of doses. [36] To induce a level of

UVB exposure that is similar to medium level sunburn in a human, a quantity

of UV radiation (A and B) standardized to the UVB (270 mJ/cm2) component

is applied. Even with relatively mild sunburn, a vast number of changes in skin

physiology and structure are induced that could affect NP penetration. For ex-

ample, within the first few hours, prostaglandin synthesis can be observed. [37–39]

Prostaglandins target the E-cadherin regulating receptors such that within hours

E-cadherin levels are significantly decreased. [36] Expression of tight junction re-

lated proteins (ZO-1, Claudin-1, and Occludin) are also perturbed following UVB

exposure. [40] These proteins are important in considering NP penetration through

UVB exposed skin, as they promote intercellular adhesions. Loosening of these

adhesions allows for the corresponding cellular proliferative response to quickly

replenish differentiating epidermal cells that form a thickened stratum corneum

layer. [41] While this UVB repair process initiates, NP may encounter loosened

intercellular adhesions causing an outside-in defect that could favor penetration.

Alternatively, one can imagine that the accelerated keratinocyte proliferation and

differentiation response enhances the net migration of cells to the skin surface

which may help prevent NP translocation. Clearly, the competing processes of


loosened intercellular junctions, oxidative damage, and the hyperplasia response

of the epidermis following exposure to a damaging UVB dose all provide potential

mechanisms to impact the permeability of NP through skin. The in vivo model

presented in this chapter comprises initial efforts to assess these effects in a qual-

itative fashion. This model greatly advances the current state as it eliminates the

need for keeping ex vivo tissue viable during the experimental course and allows

normal progression of the UVB inflammatory and hyperplasia responses and other

skin barrier effects to be experimental variables.

2.2 Materials and Methods

2.2.1 QDs and Vehicle Preparation

To explore the effects of NP skin penetration using an in vivo model, QDs were

prepared in a vehicle appropriate for skin application and quantified its material

properties. A key challenge in designing in vivo experiments is that it is not pos-

sible to have a large volume of application fluid or to protect the application area

to keep fluid from evaporating, as the animal is still mobile and occlusion of the

skin significantly affects barrier function. [42] In this chapter, we chose an appli-

cation vehicle containing 75% glycerol mixed with 25% carboxy QD (Invitrogen

ITK 565 nm emission) stock solution (pH = 9.0 borate buffer, 8 μM QDs). We

chose glycerol as the diluent for several reasons. It is present in many commer-

cial cosmetics; it is viscous, allowing it to be spread evenly on the backs of the

mice without dripping off; it is of a similar pH to the surface of the skin (pH ∼

5.0-6.0), [43] which will prevent any pH driven barrier effects; and it is hygroscopic,

preventing quick drying that would cause QD clumping and affect penetration. To


ensure that glycerol did not negatively affect the stability of the QD solution, we

made particle size measurements using a Malvern Instruments Zetasizer Nano ZS

(Malvern Instruments Ltd., Worcestershire, United Kingdom) without sonication

2 and 24 h after solution formulation with room temperature storage.

2.2.2 QD Application to Mice

To examine the skin penetration of QDs, we used 6-7 week old SKH-1 wild-type

mice (Charles Laboratory) backcrossed for seven generations with C57B16/SV129

to create congenic hairless albino mice weighing 25–30 g. Mice were allowed access

to water and standard mouse feed ad libitum and housed under constant humidity

and temperature with 1 mouse per cage and 12 h light/dark cycles during QD

experiments. Our experimental design used n =2 mice at each exposure condition

(UVB exposed and unexposed) and at each time point (8 and 24 h after QD

application). Prior to application of the QD glycerol solution each mouse was

fitted with an Elizabethan collar (Braintree Scientific, Braintree, MA) to prevent

removal and ingestion of applied QDs. Each mouse was treated with 10 µL of the

prepared QD solution applied over a ∼6 cm2 area of their back. This provided a

final QD dose of ∼3 pmol/ cm2. The collars minimally affected mouse behavior,

with the only effect being a slightly more subdued activity level. Mice were

sacrificed at two time points (8 and 24 h) following QD application. All procedures

were approved by the Institutional Laboratory Animal Care and Use Committee

of the University of Rochester Medical Center.


2.2.3 UVB Radiation Protocol

Half of the mice in this experimental design were treated with a single UVB dose.

During UVB exposure mice were housed one per cage in a standard laboratory

setting according to procedures detailed elsewhere. [37] Briefly, mice were exposed

at a distance of 15 in to UVA Sun 340 lamps, which emit across the UVA (320-400

nm) and UVB spectra (290-320 nm). The lamps were calibrated to UVB output

using an IL1700 light meter (International Light) with a SED 240 probe (255-320

nm detection). The total dose was calculated using the measured value and length

of exposure. For our experiments, the irradiated mice were exposed to an acute

dose of 270 mJ/cm2 UVB. Within the 24 h time frame of this experiment, mice

do not exhibit obvious signs of sun damage on their skin. Mice living longer (3-5

days post UVB exposure) do however develop a noticeable erythematous response

on their backs (Figure B.2, Appendix B). [37] The QD solution is applied to UVB

treated mice immediately (within 1 h) according the procedure described above.

The QD solution is also applied to the non-UVB treated mice (control) at the

same time.

2.2.4 Skin Tissue Cryo-Processing

After sacrificing the mice, tissue was harvested using several techniques. A portion

of the skin was snap frozen using liquid nitrogen and stored at -80 C. These

samples were processed for analysis by mounting the skin in TEK OCT (Sakura

FineTek USA Inc. Torrance, CA). Skin was sliced onto a microscope slide using

a Microm HM 525 cryostat (Mikron Instruments, Inc. San Marcos, CA) at 10

µm thickness, with the blade changed between slices and slicing from the dermis

to epidermis. The blade precautions were taken to avoid accidental transfer of


QDs from the skin surface to epidermal and dermal layers when slicing. After

slicing, all frozen sections were fixed in 5% formalin in PBS for 10 min. They were

then stained lightly with Gill’s Hematoxylin and mounted using Vectashield DAPI

mounting media (Vector Laboratories, Inc. Bulingame, CA). To help highlight the

stratum corneum, frozen sections fixed in 5% formalin were blocked using CAS-

BLOCK (Invitrogen Corp. Carlsbad, CA) and incubated with rabbit anti-mouse

Loricrin antibody (Covance, Berkeley, CA) for 90 min. After washing with PBS, a

goat anti-rabbit Texas Red secondary antibody (Rockland Immunochemicals, Inc.

Gilbertsville, PA) was then applied for 90 min to develop the color. Loricrin was

chosen because it is a very late releasing protein in the stratum granulosum that

is present in large quantities in the stratum corneum. [44] The slices were analyzed

under fluorescence microscopy (Nikon Eclipse E800 with a Spot RTS camera)

with a long-pass filter (355-365 nm excitation and emission of 420 nm and up) for

collection and penetration trends. To help evaluate the effect of UVB exposure

on QD skin penetration, we measured the thickness of the stratum corneum using

Spot Advance software. For each mouse, two different slices were photographed

in three locations at 40x magnification. Each was then measured five times,

giving a total of n=60 for each treatment condition. A three-way ANOVA test

was performed using the MATLAB statistics toolbox with a confidence level of

99%. Data collection was randomized to ensure no bias was introduced by the

experimenter.

2.2.5 Confocal Microscopy

To provide support for our fluorescent microscopy findings, further examination

of the QD skin penetration profiles was performed using fluorescence confocal


0

5

10

15

20

25

30

35

40

45

50

Dia

met

er (n

m)

Carboxyl QDots in75% Glycerol

Carboxyl QDots inddH2O

Carboxyl QDots in75% Glycerol Sonicated

Figure 2.1: QD SizeThe relative size of quantum dot nanoparticles as measured using a Malvern Zeta-sizer. All measurements are the average of 6 measurements per sample. The 75%glycerol QDs show a larger size, but the average remains similar over the timecourse of our experiment (24 hours), with a small increase in the distribution ofthe peak width (Fig. S1). The size in 75% glycerol solution compares well withthat in deionized (DI) water.


microscopy. Whole skin samples were mounted in a Mowiol 4-88 (Sigma 81381)

mounting media containing glycerol before imaging using an upright DMRE Leica

TCS SP Spectral Confocal Microscope equipped with Stereo-Investigator software

(MBF Bioscience, Williston, VT) and excitation from a 488 nm argon laser. The

tissue from each sample was imaged by differential interference microscopy (DIC)

and with a narrow fluorescence window (555-585 nm) to image target QD emission

(∼565 nm) only. Acquisition of 0.5 µm confocal slices was taken through the

thickness of the skin samples (∼25 µm) using the Stereo-Investigator software.

Three-dimensional stack reconstruction and image processing was performed using

ImageJ software (NIH).


Transmission electron microscopy (TEM) was used to examine ultrastructural de-

tails of QD nanoparticle skin penetration through the skin and their end locations

at the cellular level. After 24 h fixation in 2.5% glutaraldehyde, the whole skin

samples were postfixed in osmium tetroxide and silver enhanced using a standard

AURION R-GENT SE-EM reagent and protocol. The silver enhancement selec-

tively deposited on the QDs to allow them to be distinguished easily from the

surrounding tissue. After silver enhancement of the QDs, the skin samples were

dehydrated using graded alcohol baths (25%, 50%, 75%, and 100%) and then in-

filtrated with and embedded in Spurr epoxy resin with overnight polymerization

at 70 C. After embedding, the samples were cut to 1-2 µm with a glass blade and

finally sliced at 70 nm with a diamond knife and placed on copper grids. Nanopar-

ticle localization was evaluated using a Hitachi 5100 TEM apparatus with EDAX

attachment to provide elemental analysis spectra of samples. To ensure silver en-


hancement only worked on QDs, a negative control was processed from a mouse

treated with glycerol only. After silver enhancement, dehydration, epoxy mount-

ing, and slicing, measurements were performed on the negative control sample (no

QDs) using equivalent procedures as with QD exposed skin samples.

2.3 Results and Discussion

2.3.1 Application Vehicle Characterization

In designing our experiment, the first concern was formulation of an application

vehicle and demonstration that QD size stability was unaffected. Glycerol was

identified as a potential diluent. Particle size analysis, summarized in Figure 2.1

(raw data Figure A.1, Appendix A), revealed that QDs prepared in deionized

(DI) water and in 75% glycerol remain monodispersed exhibiting a particle size of

∼20 and ∼33 nm, respectively. QDs formulated in DI water and glycerol remain

stable after storing the solution at room temperature for at least 24 h. Zeta

potential measurements in DI water suggest a weak negative surface charge of

approximately -20 mV. Surface charge measurements in 75% glycerol could not

be made due to instrumentation limitations resulting from high solution viscosity.

However, zeta potential measurements made at lower glycerol levels (25% and

50%) suggest the QDs retain their intrinsic negative surface charge as measured

in DI water. The hygroscopic properties and pH matching of glycerol to the

stratum corneum [43,45] (pH ∼ 5.0-6.0) combined with a stable particle size over

the time period of interest (24 h) suggested glycerol as an ideal QD application

vehicle for our in vivo experiments. This vehicle enabled application of a small

volume of fluid that was easily spread over the application area and provided


0.0

5.0

10.0

15.0

20.0

25.0

30.0

Thic

knes

s (µ

m)

8hr Ctrl Mouse 8hr UV Mouse 24hr Ctrl Mouse 24hr UV Mouse

****

**

Figure 2.2: Stratum Corneum ThickeningThe stratum corneum shows thickening as the keratinocytes quickly differentiateinto mature corneocytes in response to UVB light exposure. The differentiationresponse- as measured by stratum corneum thickness- increases with time afterUVB exposure. The unexposed mice do not show a thickening response despitepresence of the QD borate buffer/ glycerol solution. Each of the UVB exposedconditions demonstrate significant difference from each other and the controlsusing Student’s unpaired t test (**, p<0.01).


humectant properties, preventing rapid evaporation.

2.3.2 Validation of UVB Induced Skin Response

After processing skin tissue with the methods described, hematoxylin stained cryo-

preserved tissue sections were examined under bright field optical microscopy to

validate evidence for the biological effects of exposure to UVB in our model sys-

tem. It is known that UVB exposure to skin affects keratinocyte differentiation

and cell division in the epidermis. This UVB induced skin repair response causes

a rapid increase in epidermal proliferation and differentiation events with a re-

sultant thickening of the stratum corneum and hyperplasia of the epidermis that

initiates within hours after UVB exposure and remains elevated for more than

3 days. [39] Validation of these responses in our model system after just 24 h is

important as epidermal remodeling may potentiate an outside-in barrier defect

allowing for enhanced QD NP skin penetration. In this chapter, we used stratum

corneum (SC) thickening as a metric. Results confirm the induction of the UVB

repair response as stratum corneum thickening was clearly observable after only

8 h (Figure 2.2). After 24 h, the difference in stratum corneum thickness between

the unexposed and the UVB exposed samples was pronounced, increasing from

∼9.7 to 18.9 µm, respectively. It is interesting to note that there is a statistically

significant difference between the 8 h UVB exposed and the 24 h UVB exposed

samples, validating that the UVB induced keratinocyte proliferation and differen-

tiation response had initiated. It is also important to note that ANOVA testing

criteria at a 1% confidence level found no observable difference between the 8 and

24 h non-UVB exposed samples. This is of note as it suggests that the applied

QD mixture did not affect the stratum corneum thickness and therefore can be


iii. 8hr Ctrlii. 24hr UVi. 8hr Ctrl

A

B

i. 8hr Ctrl ii. 24hr UV iii. 8hr Ctrl

Figure 2.3: QD Collection Trends(A) The common collection pattern of QDs in skin defects and folds observedthroughout the samples. The weaknesses in the stratum corneum that are presentin many of these cases may contribute to increased stratum corneum penetrationpossibility. (B) Three examples of the collection pattern commonly occurring inthe mouse hair follicles. (i) and (ii) are stained with DAPI blue and (iii) is stainedwith a combination of DAPI and Texas Red Loricrin antibody, a protein found inhigh abundance in the stratum corneum. The greenish/yellow spots are the QDs.


thought to have a minimal effect on keratinocyte proliferation and differentiation.

Histological studies (described below) found no evidence for immune infiltration

in the non-UVB exposed samples, further suggesting a negligible effect of QD

application to non-UVB exposed skin over the studied time course. In contrast

to the SC, epidermal thickening was not as pronounced in the 24 h time period

(Figure A.2, Appendix A). This is consistent with expectation, as the UVB in-

duced hyperkeratosis (stratum corneum thickening) response has been shown to

increase more quickly than the hyperplasia response (epidermal thickening). [41,46]

Analysis of skin samples 4.5 days post UVB exposure (Figure A.3, Appendix A)

confirm the progression of gross morphological changes in both the epidermis and

the stratum corneum. The ability to quantifiably measure physical evidence of

the biological response to UVB exposure provides evidence of a UVB induced

repair process that includes down regulation of E-cadherins [36] and alteration in

the expression of tight junction proteins. [40] It is of interest to investigate how

the competing effects of loosened cell junctions and accelerated cellular prolifer-

ation, differentiation, and migration toward the skin surface combine to impact

NP penetration.

2.3.3 QD Penetration: Effect of UVB

To examine QD penetration, the histological slices provided important insights.

Results consistently found a similar trend of increased penetration for both treat-

ment conditions (8 and 24 h) with UVB. Most strikingly, under no circumstances

is there evidence for massive QD penetration, even for UVB exposed mice 24 h

after QD application. Our data consistently find that QDs preferentially collect

in folds and defects in the stratum corneum (Figure 2.3A, Figure A.4 Appendix


Figure 2.4: Hair Follicle QD CollectionA confocal image taken at 40x magnification. The top image is the differentialinterference contrast (DIC) image that shows twin hairs going under the surfaceof the skin. The bottom image is the same field of view with a 555-585 nmfluorescence window to illuminate the QDs. The side and bottom profile the 3-dimensional stack of images (fluorescence into the skin), giving a clear view of thedepth and hair follicle location of a large quantity of QDs.


A) as well as in hair follicles (Figure 2.3B). The collection effect is seen even in

the non-UVB exposed mice. Underneath the folds and defects, there are often

fewer stratum corneum layers and a thinner epidermal layer, which could poten-

tially increase long-term risk of penetration at these sites. The collection of NP

in folds, defects, and hair follicles is a well-documented phenomenon [47,48] that

is specific to NP in the size range used in our experiments, and may contribute

to increased penetration over time and provide an opportunity for targeted drug

delivery applications through the follicle. [47,49,50]

To provide further evidence of this occurrence, we used whole tissue fluores-

cence confocal imaging. The confocal imaging provided us with a clear three-

dimensional perspective on the collection phenomenon, as it showed a profile with

QDs collecting all the way into the lower portions of the hair shaft (Figure 2.4).

The confocal imaging also serves to highlight the much larger proportion of QDs

available in folds/defects and especially the hair follicle.

Despite the similarities in trends throughout the different time and UVB ex-

posure conditions, there were some distinct differences between the samples in

terms of penetration levels. To explore the differences in mouse skin penetration

between the time points and the UVB exposures, we first considered the cryo-

preserved tissue sections. It is important to reiterate that none of the penetration

observed was at a very high level, but we were able to observe increased pene-

tration for the UVB as compared to the control at both the 8 h and the 24 h

time points (Figure 2.5). The 8 h UVB exposed samples showed a higher rate

of penetration over the 8 h control. This was seen in a variety of structures, but

appears most commonly in areas that have a defect in the stratum corneum or by

hair follicles (Figure 2.5B). This trend is mirrored at the 24 h time point as well.

The difference between the 8 h UV and the 24 h UV mice is not as marked. The


A

B

iii. 8hr Ctrl

ii. 24hr UV

i. 8hr Ctrl

i. 24hr UV

ii. 24hr Ctrl

Figure 2.5: UVB Induced QD Skin Penetration(A) An overview (20x magnification) of the 8hr ctrl (i) and 24hr ctrl (ii). Minimalpresence of QDs can be seen even in the lower stratum corneum layers. (B)Example slices of the 24hr UVB exposed mouse skin with high penetration areasin the dermis highlighted by magnified insets.


deepest levels that showed evidence of QDs was an additional point of interest.

Presence into the stratum corneum could be observed throughout all skin samples

(Figure 2.3A). In a few isolated locations for the non-UV exposed mice, some

of the outermost keratinocytes demonstrated QD presence in what appeared to

be perinuclear locations (Figure 2.5A,ii). However, the presence was far greater

throughout the UV exposed mice, which showed additional QD presence much

deeper in the tissue. Tissue levels as deep as the dermis were common but still at

low levels. Common trends and a more detailed description of these instances will

be shown in future work which will seek a greater understanding of the relative

importance of para- and trans-cellular transport mechanisms and identify differ-

ences that may result from a change in application conditions, such as more severe

UVB damage or application of QDs at a longer time point after UVB exposure.

2.3.4 QD Penetration: Mechanistic Insight

To provide mechanistic insight into the penetration pathway taken by QDs to

breach the stratum corneum barrier, TEM with silver enhancement was used

(Figure 2.6). The silver enhancement procedure increases QD particle size to

∼35-45 nm (Figure A.5, Appendix A) and allows clear imaging of them against

the tissue background. EDAX was used to determine the elemental composition of

the spots believed to be silver enhanced QDs and found a strong presence of silver

in all cases of spots believed to be NP (Figure 2.7). TEM provided convincing

evidence that the QDs were getting through the stratum corneum through the

intercellular lipid lamellae along the edges of differentiated corneocytes (Figure

2.6A,B). To confirm that silver enhancement was selective for the QDs, a control

mouse (24 h UVB but no QDs, 75% glycerol only) was examined (Figure 2.6D) and


A

C D

B

Figure 2.6: QD Penetration PathwayTEM imaging of 24hr UVB exposed mouse skin sections. (A) The penetrationpathway through the skin can be clearly seen, and is shown in more detail in (B)with the large dark spots being the NP. (C) Another section of skin demonstratingthe penetration pathway and with an example silver enhanced QDs present inthe epidermal layer. (D) The negative control (no QDs, glycerol only) of silverenhanced mouse skin 24 hr UVB exposure.


spectra taken of suspicious debris (Figure A.6, Appendix A). No presence of silver

could be found in any of the suspicious dark spots on non-QD control samples.

It is our observation from analysis of a number of the experimental tissue slices

that silver enhanced QDs are more frequently seen in viable epidermal layers such

as the stratum granulosum (Figure 2.6C) using TEM compared to cryo-histology

analysis. There were also a number of QDs present deeper in the dermis that were

taken up by various cell types and dermal structures.

2.4 Conclusions

These studies demonstrate the importance of skin condition to affect the pene-

tration of QD nanoparticles (∼30 nm diam.) in the in vivo SKH-1 mouse model.

We have shown that QDs work their way between the corneocytes of the stratum

corneum and penetrate deep in the epidermis and dermis of an in vivo model with

UVB penetration exacerbation. This is an important advancement as it suggests

that UVB induces an outside-in barrier defect likely due to a loss of epidermal cal-

cium gradient and resultant stratum corneum lipid disruption, [51,52] and through

loosening of cell-cell junctions that down regulate following an acute UVB expo-

sure. The accelerated epidermal proliferation and differentiation UVB skin repair

response is insufficient to prevent QDs from breaching the skin barrier. It is im-

portant to note that the penetration of QDs did not attain the drastic levels seen

in some ex vivo nanoparticle tissue studies [53] even with mechanical flexing asso-

ciated with normal animal motion. [54] The collection of NP in folds, defects, and

hair follicles provides a possible mechanism for transport when taken together

with the barrier function disruption and stratum corneum intercellular weaken-

ing effect of UVB. [36] We believe that the QD penetration is strongly dependent


A

C

B

Figure 2.7: QD EDAX ConfirmationTEM analysis of 24 hour UVB exposed mouse skin with EDAX evaluation ofelemental composition. The quantum dot components are unable to be detected,but with selective silver enhancement, silver peaks are clearly visible in the spectra.(A¬) A lower magnification view showing the presence of a couple silver enhancedQD clusters in the stratum granulosum. (B) A higher magnification view of thelarger cluster of silver enhanced QDs. (C) The EDAX spectrum for the clustershown in (B), demonstrating a strong presence of silver, with additional presenceof copper and nickel that are elements present in the TEM grid used.


on the condition of the skin and the characteristics of the QD (size and surface

chemistry). This is an important discovery for nanoparticle safety concerns as

consumers often apply sunscreens containing metal oxide nanoparticles of similar

size and raw material properties to UV-exposed skin. The minimal QD penetra-

tion observed in our study on barrier intact (non-UVB exposed) skin supports the

preponderance of current literature suggesting TiO2 and ZnO NP used in commer-

cial sunscreens exhibit limited penetration in layers below the lower SC. [13,55] The

increased QD penetration seen with UVB damage raises concern, but this result

does not directly address the issue of metal oxide NP penetration through UVB

damaged skin. A wide variety of surface coatings on TiO2 and ZnO NP are used

in formulating commercial sunscreens to make them waterproof and to improve

their application characteristics. These coatings may alter their skin penetration

characteristics. The next chapters of this thesis build on this work to quantify the

amount of QD penetration and investigate the effects of UVB on QD interaction

at the cellular level.


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96

Chapter 3

UVB and Delayed QD Skin

Permeability

Quantification of QD skin penetration riskwith UVB induced delayed barrier impair-ment as measured by TEWL1

1Portions of this chapter are adapted from Mortensen, L.J., Gelein, R., Bentley, K.L.D.M.,Elder, A., and DeLouise, L.A. Quantification of quantum dot skin penetration risk with UVBbarrier impairment as measured by transepidermal water loss, submitted to Toxicol Sci 2011.

CHAPTER 3. UVB AND DELAYED QD SKIN PERMEABILITY 97

3.1 Introduction

This chapter endeavors to quantify both the barrier disruption effects of UVB and

the skin penetration of quantum dots (QDs). QD skin penetration is an impor-

tant topic, as established by the previous chapters in this thesis, and nanoparticle

research as a whole is growing, with the National Nanotechnology Initiative es-

timating that the United States federally funded $1.5 billion in research for the

2009 fiscal year, [1] and over 1000 consumer products available in 2009 as listed by

the Project on Emerging Nanotechnologies. [2]

Work presented in the previous chapter was able to discern minimal skin pen-

etration of QDs with intact skin barrier, with a qualitative increase found in skin

penetration after UVB exposure. These findings correspond well to other qualita-

tive literature investigating the skin penetration of QDs in intact [3,4] and barrier

disrupted skin. [4] Qualitative understanding of NP skin penetration yields impor-

tant information, but to allow comparison of multiple conditions and more subtle

barrier disruption, quantitative techniques are a necessity. To this end, work in

our lab has found low levels of QD skin penetration in healthy ex vivo human skin

that can be observed to increase with exposure to thioglycolate depilatory agent

or tape stripping by flow cytometry. [5] Work by Gopee et al. has found a systemic

increase in skin penetration of QDs after dermal abrasion with quantification us-

ing mass spectrometry. [6] Lopez et al. were able to observe low levels of QD skin

penetration that was increased using ultrasound and sodium lauryl sulfate treat-

ment using mass spectroscopy of a separated dermis. [7] Of these techniques, the

most applicable to the impact of UVB on QD skin penetration in an in vivo model

is that of organ collection and mass spectroscopic analysis, which will be used in

this portion of this dissertation.


The effect of UVB on skin barrier found in the previous chapter is built upon

in these subsequent experiments. While UVB does have an immediate impact on

the skin, the most notable barrier disruption occurs several days after exposure.

The UVB wavelengths are strongly absorbed by epidermal DNA, which causes

the formation of pyrimidine dimers and a subsequent flood of signaling molecules,

including cytokines and prostaglandins. [8,9] Soon thereafter, a strong increase in

DNA repair processes and DNA synthesis occurs as a result of the acute dam-

age via the p53 and COX-2 pathways as the basal keratinocytes begin to rapidly

proliferate. [10–13] Corresponding to this increase is a prostaglandin-driven decrease

in the E-cadherins junctions, which allows the newly proliferated cells to migrate

quickly to the stratum corneum surface. [14] A strong barrier disruption as mea-

sured by transepidermal water loss (TEWL) occurs after DNA synthesis, about

three days after UVB exposure and reaches a peak within the next 1-2 days [12,15,16]

with a similar magnitude as the treatment of skin with common barrier disrupting

agents (i.e. acetone, tape stripping, and surfactant treatments). [17,18] This inside-

out water loss defect has been demonstrated in an early study to increase the

outside-in skin penetration of small molecules such as hydrocortisone in vivo. [19]

The mechanism of UVB barrier abrogation is not completely clear, but has been

suggested to be due to a immune cell mediated pathway [12] and is correlated to

a loss of the epidermal calcium gradient (also important for the cadherins junc-

tions), [20] a disorganization of stratum corneum lipids, [16] and the appearance of

inadequately differentiated cells in the stratum corneum (i.e. the appearance of

nuclei). [21] After 7-8 days, the last of the damaged keratinocytes are sloughed off

and skin barrier status returns to normal. The possibility of exposure to UVB

and NPs over the time period which has a substantial TEWL barrier defect sug-

gests the need for research investigating this combination, which is a focus of this


study along with investigation of the validity of TEWL for prediction of NP skin

penetration.

TEWL is a well established technique that functions by measuring the flux

of water vapor out of the skin over a controlled area. A number of studies have

detailed the correlation between TEWL measurements and permeability of skin

compromised by chemical means (such as sodium lauryl sulfate [22] or acetone [17]),

by physical means (such as tape stripping or skin surface biopsy [23]), or by means

of UVR exposure. [12,16,20,21] Some researchers have gone further, and have corre-

lated TEWL measurements with the permeability of skin to small molecules based

on hydrophilicity. [17,24,25] No studies, however, have examined the relationship be-

tween defective skin barrier function as measured by TEWL and skin permeability

to NPs. Previous work using increasing molecular weight PEG molecules (300 Da,

600 Da, and 1000 Da) suggests that with a given barrier disruption such as tape

stripping, TEWL can predict the permeation behavior of large PEG molecules, [18]

but this has not been extended to the size range of NPs. This chapter uses a

designed experiment approach to evaluate the impact of UVB on skin barrier

function in the in vivo SKH-1 mouse model at a variety of doses and endeavors

to relate this to the penetration of carboxylated QDs using microscopy and quan-

titative elemental organ analysis determine skin penetration. Our study fills an

important void in the literature and suggests a path forward to evaluating NP

skin penetration risk.



3.2.1 QD Functionalization and Vehicle Preparation

To prepare carboxylic acid coated QDs, we purchased CdSe/ZnS core-shell quan-

tum dots (NN-Labs) with a peak emission wavelength of 620 nm in the organic sol-

vent toluene. To allow use in a biological environment, we prepared aqueous QDs

by ligand exchange with dihydrolipoic acid (DHLA) as described previously. [15]

Briefly, a ∼10,000x molar excess of pure DHLA is added to 1 mL methanol and

the pH adjusted to 11.0-12.0 using tetramethylammonium hydroxide pentahydrate

powder (Sigma-Aldrich Inc.). The QDs (250 µL) are precipitated from toluene

using excess methanol/acetone (50/50) and centrifugation at 12,000 rpm. They

are then resuspended in tetrahydrofuran (THF) and added dropwise to the DHLA

reaction mixture. The reaction is incubated at 60C with stirring for 3 hours, and

at room temperature overnight. The QDs are mixed with excess ether to pre-

cipitate and centrifuged at 12,000 rpm for 10 min. The ether is poured off, the

pellet dried with nitrogen gas, and the QDs resuspended in water. The QDs are

dialyzed using a 5kD molecular weight cutoff DispoDialyzer filter (Harvard Ap-

paratus Inc.) and 500x excess volume of water for 72 hours with water changing

every 24 hours. After dialyzing, the concentration is determined by measuring

the absorption at the first exciton and using an extinction coefficient from the lit-

erature with Lambert-Beer’s law. [26] To determine stability of our functionalized

QD, hydrodynamic diameter and zeta potential were measured in water and the

in vivo application vehicle (described below) using a Malvern Instruments Zeta-

sizer Nano ZS (Malvern Instruments Ltd, Worcestershire, United Kingdom) at a

concentration of ∼10-20 nM.


3.2.2 UVB Irradiation and TEWL

To examine barrier response by TEWL and skin penetration of QDs, we used

SKH-1 wild-type backcrossed mice developed as described previously (Chapter 2).

Mice were allowed access to water and standard mouse feed ad libitum and housed

under constant humidity and temperature with 1 per cage and 12 hour light/dark

cycles during UV-irradiation and recovery experiments. All mouse experiments

were performed on animals 8-10 weeks in age weighing 25-30 g. For the TEWL

experiments, a designed experiment approach was utilized with a full factorial 9

treatment condition array with a 2-level treatment condition gender factor (male

and female) and a 4-level treatment condition UVB dose factor (0 mJ/cm2, 180

mJ/cm2, 270 mJ/cm2, and 360 mJ/cm2) that was repeated twice. TEWL was

measured using the Tewameter TM 300 with a mouse adaptor (Courage-Khazaka)

with 30-60 seconds equilibration time to ensure consistent measurement. The

TEWL was measured for 4 days to establish a baseline. The mice were irradiated

one per cage in a standard laboratory setting according to procedures detailed

previously (Chapter 2). Briefly, mice were exposed at a distance of 15 inches

to UVA Sun 340 lamps, which emit across the UVA (320-400 nm) and UVB

spectra (290-320 nm). The lamps were calibrated to UVB output using an IL1700

light meter (International Light) with a SED 240 probe (255-320 nm detection).

The total dose was calculated using the measured value and length of exposure.

Their TEWL was then measured immediately after UVB exposure and each day

subsequently for 7 days at the same time each day to limit circadian rhythm error.


3.2.3 QD Application to Mice

For QD application, a 2-factor 2-level experimental design was used with n=4.

The factors selected were UVB (either no UVB exposure or 360 mJ/cm2 UVB

irradiation) and QD application (vehicle only or QDs in vehicle). To attempt

to achieve the highest levels of QD penetration possible, the skin was exposed

to QDs or vehicle 3.5-4.5 days post UVB exposure using the previously discussed

technique (Chapter 2). Prior to application of the QD glycerol solution each mouse

was fitted with an Elizabethan collar (Brain tree Scientific) to prevent removal and

ingestion of applied QDs. Each mouse was treated with 30 µL of 3.5 µM DHLA

QDs in 30% glycerol over a ∼6 cm2 area of their back. This provided a final QD

dose of ∼17.5 pmol/cm2. The collars minimally affected mouse behavior, with a

slightly more subdued activity level being the only observable impact. Mice were

sacrificed 24 hrs following QD application. After sacrifice, the skin application

area was harvested from the back of the mouse and treated as described below.

All procedures were approved by the Institutional Laboratory Animal Care and

Use Committee of the University of Rochester Medical Center (2010-024/100360).

3.2.4 Dissection and Organ Analysis

After sacrifice, excess QDs remaining on the skin surface were carefully wiped

off using sterile gauze and 1x PBS and dried. The proximal lymph nodes (ax-

illary and brachial) and the liver were harvested from each animal for analysis

and weighed. Organs were harvested using dedicated instruments, with the con-

trol animals being dissected first. Between each animal, the dissection surface

covering was changed and the instruments cleaned and rinsed in H2O, acetone,

sonicated for 10 minutes in 1% HNO3, and rinsed again to remove residual acid


and eliminate potential contaminants. The tissues were placed directly into di-

gestion vials and wet ashed with ultrapure 70% nitric acid (Baseline, SeaStar

Chemicals Inc., Sidney, British Columbia, Canada). After ashing, the tissue

residue was resuspended in HNO3 and the concentration was adjusted to 2%

with 18 M Ω deionized water before graphite furnace atomic absorption spec-

troscopy analysis. Quantification was achieved through comparison to reference

standards (Standard Reference Material 1577b from bovine liver; National Insti-

tute of Standards and Technology, Gaithersburg, MD). The Cd limit of detection

(LOD) was 0.033 ng/mL and the limit of quantification (LOQ) was 0.111 ng/mL.

This equated to LOD=0.176±0.002 ng and LOQ=0.588±0.006 ng in the liver,

and LOD=0.071±0.0002 ng and LOQ=0.235±0.0006 ng in the lymph nodes. To

ensure that experimental error did not contribute to the Cd signal found in 24 h

QD applied liver and lymph tissue, a 0 h experimental control was executed. For

this control, non-irradiated animals had the standard concentration and volume

of QDs applied and were immediately sacrificed and Cd organ distribution evalu-

ated. To calculate percent of applied dose, the amount of Cd in the applied dose

was measured as a control. Statistical analysis was performed as described below.

3.2.5 Skin Tissue Cryo-Processing

A portion of the skin was snap frozen using liquid nitrogen and stored at -80C.

These samples were processed for analysis by mounting the skin in TEK OCT

(Sakura FineTek USA Inc. Torrance, CA). Skin was sliced onto a microscope slide

using a Microm HM 525 cryostat (Mikron Instruments, Inc. San Marcos, CA) at

10 µm thickness, with the blade changed between slices and slicing from the dermis

to epidermis. The blade precautions were taken to avoid accidental transfer of QDs


from the skin surface to epidermal and dermal layers when slicing. After slicing, all

frozen sections were fixed in 5% formalin in PBS for 10 minutes and mounted using

Vectashield mounting media (Vector Laboratories, Inc.). The slices were imaged

under a wide field fluorescence microscope (Olympus IX70 with QImaging Retiga

EXL camera) with a mercury lamp excitation source (360/30 bandpass filter)

and narrow emission (620/10 bandpass filter), and phase contrast microscopy to

allow visualization of skin structures while minimizing autofluorescence in the

QD fluorescence images. Image processing was performed using ImageJ, with

fluorescent images processed equivalently and reported in red fluorescence and

with an ImageJ look up table (LUT) applied to highlight penetration differences.


Transmission electron microscopy (TEM) was used to examine ultrastructural

details of QD skin penetration and end locations at the cellular level. After 24

hour fixation in 2.5% glutaraldehyde, the whole skin samples were postfixed in

osmium tetroxide and silver enhanced using a standard AURION R-GENT SE-

EM reagent and protocol. The silver enhancement selectively deposited on the

QDs to allow them to be distinguished easily from the surrounding tissue. After

silver enhancement of the QDs, the skin samples were dehydrated using graded

alcohol baths (25%, 50%, 75% and 100%) and then infiltrated with and embedded

in Spurr epoxy resin with overnight polymerization at 70C. After embedding,

the samples were cut to 1-2 µm with a glass blade and finally sliced at 70nm

with a diamond knife and placed on copper grids. The nanoparticle localization

was evaluated using the Hitachi 5100 TEM apparatus with EDAX attachment

to provide elemental analysis spectra of samples. To ensure silver enhancement


was optimized for contrast of QDs, negative controls were processed from vehicle

only treated mice. After silver enhancement, dehydration, epoxy mounting, and

slicing, measurements were performed on the negative control sample (no QDs)

using equivalent procedures as with QDs exposed skin samples.

3.2.7 Statistical Analysis

Experimental planning and statistical evaluation using the ANOVA technique was

used for the experiments presented in this study. For TEWL, a 2-way ANOVA

was used to evaluate the significance of UVB dose and gender on the TEWL re-

sponse at each day with n=2, with group comparisons performed using Students

paired or unpaired t-tests. For organ distribution of Cd, n=4 was used. In the

liver, statistical analysis of total Cd organ distribution was performed using 2-

way ANOVA analysis. For lymph tissue, a 2-way ANOVA experimental plan was

implemented to evaluate total Cd distribution. However, the non-QD exposed

lymph tissues all contained Cd levels that were less than the limit of quantifi-

cation or detection, which eliminated the possibility of ANOVA comparison. To

compare the individual groups in liver and lymph, Students unpaired t-tests were

performed.

3.3 Results

3.3.1 TEWL

For these experiments, the TEWL of the mice was recorded at each day for 4 days

to establish a baseline. The relative humidity and temperature of the room were

kept as constant as possible, but their variability provided one of the important


***

*

*** ******

***

***** ***

0.5

1.5

2.5

3.5

4.5

5.5

6.5

7.5

8.5

9.5

10.5

0 1 2 3 4 5 6 7Time (Days after Irradiation)

Nor

mal

ized

TEW

L

0 mJ/cm2 UVB180 mJ/cm2 UVB270 mJ/cm2 UVB360 mJ/cm2 UVB

******

***

Figure 3.1: Impact of UVB on TEWLThe effect of UVB on skin barrier function as measured by TEWL. Increasingdoses of UVB exposure increase the incurred barrier defect (from 0-360 mJ/cm2).A statistically significant increase is observed with all UVB doses with peaks rang-ing from days 3-6 post-UVB exposure. For this experiment, n=4 was comparedto control using Students paired t-test (*=p<0.05, **=p<0.01, ***=p<0.001).


noise contributors. This effect has been studied in greater detail by others, but in

our experiments it simply increased experimental standard deviations slightly. [27]

Additionally, each mouse demonstrated a slightly variable TEWL baseline value.

To limit the influence of inter-animal variability, the absolute baseline values for

each individual mouse were averaged together to provide a baseline, and their

TEWL after UVB exposure compared to this measurement. When the base-

line values for all mice were averaged, the absolute TEWL value was 4.15±0.73

g/m2/h. The relative TEWL values for all mice were averaged together to deter-

mine the impact of UVB on barrier function over time. After completion of the

experiment, ANOVA analysis yielded interesting results. First, analysis of means

(ANOM) was performed to show the strongest contributing factors (Figure B.1,

Appendix B). This analysis demonstrated that UVB and the day were very strong

factors, whose impact was dose and time dependent (p<0.0001 for each). Gen-

der was unimportant, suggesting that the male and female mice could be used

interchangeably in subsequent experiments (p=0.18). When the values were com-

bined a more traditional form, the chart demonstrated a 72 hour lag time before

the induction of skin barrier disruption that matched the literature. [12,16,20] The

third day after irradiation, some scaliness and redness was detected on the backs

of the mice, with measurements taken on these areas (Figure B.2, Appendix B).

The increase in TEWL was unable to be strongly detected until the appearance

of these spots of somewhat varied morphology, size, and strength on the backs

of each of the mice. Interestingly, we measured the TEWL on areas away from

the most notable UVB damage (neck and lower back) and observed no change in

the TEWL loss (Figure B.3, Appendix B). This can be attributed to the UVB

dose falloff that occurs on the sides and lower lying regions of the mice, since

irradiance decreases as the square of distance. Over the next few days, the scaly


spot was seen to gradually shrink and disappear. However, even when there was

no visible response remaining, an increase in the TEWL could still be found. Our

experimental results suggest that the amount of UVB needed to cause a signif-

icant change in skin barrier function is less than what would be substantially

noticeable on a human subject. With the dose plotted on a log scale, the TEWL

values obtained at each time point followed a linear path as suggested by an early

study, [19] and we found that slope varied depending on the day, confirming the

decreased rate of healing that occurred with high doses of UVB (data not shown).

To ease graphical interpretation, we show averages of all four mice used for each

UVB dose at each time point, with a paired Students t-test used to determine

certainty of difference over control (Figure 3.1). By 3 days post-UVB exposure,

all UVB doses generate statistically significant increase over control that varies

in the amount of time required to reach a peak, and begins to recover by 7 days

after irradiation. The TEWL achieved peak values of 2.33±0.26 fold over con-

trol for the 180 mJ/cm2 dose at 4 days post UVB exposure, 6.07±1.74 fold over

control for the 270 mJ/cm2 dose at 5 days post UVB exposure, and 8.84±0.96

fold over control for the 360 mJ/cm2 dose at 6 days post UVB exposure (Figure

3.1). When compared to the existing literature, the values observed match up

well, especially when differences in mouse strain, measuring device, and average

temperature/relative humidity are taken into account. When compared to values

obtained by tape stripping in mice, the magnitude of the change is on the order

of high barrier disruption that is known to strongly increase the penetration of

PEG molecules. [18] We expect the TEWL to be an accurate predictor of the skin

permeability to QDs, but as seen in other studies use of TEWL as a descriptor for

skin permeability relies on the specific disrupting agent and the exogenous topi-

cally applied substance. [17,18] To determine its ability to provide a prediction for


QD skin penetration in the case of UVB exposure, we next attempt to quantify

the skin penetration of QDs in mouse. The first step to enable this is to evaluate

the characteristics of our QDs in water and the application vehicle.

3.3.2 QD Characterization

To characterize our functionalized QDs, the first step was to determine particle

size and zeta potential in both water and the glycerol application solution. Us-

ing a Malvern Zetasizer, we determined the size of our DHLA 620 nm QDs to

be very similar in both deionized water and the 30% glycerol containing vehicle

(Figure 3.2). This suggests that the QDs diluted in water and glycerol maintain

a similarly monodisperse condition, with particle sizes of 14-15 nm. Zeta poten-

tial values of approximately -45 mV obtained in deionized water indicate strongly

functionalized QDs that have a large number of DHLA molecules on their surface.

The similar zeta potential values of approximately -40 mV in 30% glycerol sug-

gest that independent of solution type QDs maintain their charge and stability,

with the small differences in zeta potential being attributable to the pH difference

between the solutions. The charge stability, pH matching, and monodispersity of

our DHLA functionalized QDs in the application solution helps to maintain their

solution characteristics under our experimental conditions.

3.3.3 Fluorescence Microscopy Evaluation of QD Skin Col-

lection and Penetration

We chose to investigate the effect of UVB on QD skin penetration by applying

QDs for 24 hours 3.5 days post UVB exposure at a dose of 360 mJ/cm2. With

histological evaluation of the UVB treated vs. untreated mice, phase contrast


0.00

0.05

0.10

0.15

0.20

0.25

0.30

6 8 10 14 18 24 33 44 59 79 106 142 190 255 342 459

Dis

tribu

tion

Particle Size (nm)

QD Source Emission Peak Surface Coating Solution pH Diameter Zeta Potential

NN-Labs 620 nm DHLA Water 6.5-7.0 14±0.4 nm -45±6 mV

NN-Labs 620 nm DHLA 30% Glycerol 5.7-6.0 15±0.5 nm -40±10 mV

Figure 3.2: QD CharacterizationSize and zeta potential measurements for the DHLA encapsulated QDs used inthese experiments. DHLA surface coating enables a small stable size in water (de-picted) and the 30% glycerol application solution. The zeta potential is stronglynegative for each case, and the slight variations observed may be due to differencesin pH or viscosity.


microscopy allowed observation of a clear thickening and stratum corneum dis-

ruption due to the UVB exposure (Figure 3.3). This observation matched well

to the histological observations that would be expected based on the literature

in terms of thickening and defective differentiation. [11,21,28] When the QD pene-

tration was evaluated using histology, several observations could be made (Figure

3.3). As reported previously with 565 nm emitting QDs with slightly smaller core

sizes but similar hydrodynamic radius and surface charge, [29] with non-irradiated

skin we most commonly saw collection in the outer layers of the stratum corneum,

with some preferential collection in folds and defects of the skin, as well as along

hair follicles. On the 360 mJ/cm2 samples, similar collection trends were observed

but with a more consistent depth of penetration through the loosened intercellular

lipid lamellar structures. A number of noticeable occurrences of QDs penetrat-

ing in large quantities through the damaged stratum corneum could be found, a

representative example of which is shown (Figure 3.3). These occurrences tended

to be rare and localized with more common observation in the areas that had

the most obvious morphological damage and thickening of the stratum corneum

resulting from UVB exposure. Additionally, while instances of high penetration

areas were rare in the 360 mJ/cm2 UVB dose, they were present in some of the

slices. In contrast, QD penetration to an appreciable level was unable to be found

in non-UVB irradiated samples. This does not entirely preclude the possibility

of such occurring due to sensitivity limits of our fluorescence imaging system. [30]

Isolated instances of high fluence entry points for QDs suggest that when bar-

rier is disrupted in practice, there are “seed points” where localized weaknesses

in the barrier allow amounts of QDs to enter. Also, there is clear evidence for

the collection of QDs in the hair follicles of both control and UVB exposed mice.

These locations have been described by Paliwal et al. as lacunar pathways that


are present even in healthy skin tissue. [31] A recent study by Lopez et al. has pos-

tulated that the limited amount of QD penetration into the dermis they were able

to observe in healthy skin may have been due to the putative lacunar pathway. [7]

Investigation into the impact of chemical penetration enhancers using multiphoton

microscopy has found similar such events with skin penetration of ZnO NPs- skin

penetration of NPs appears to occur in low frequency weak or seed points that

are expanded by certain forms of barrier disruption, rather than the traditional

uniform diffusion gradient described by Fick’s law. [32] Most models of diffusion

through the skin treat barrier disruption as a uniform weakening, and as such

have limitations when it comes to the observation of a number of isolated very

low occurrence but high penetration, and models describing this behavior war-

rant investigation. Histological analysis was performed on 20-30 sections for each

treatment condition, which equates to examination of 0.02-0.03 cm2 surface area

examination area, or ∼0.3-0.5% of the application area. Although the impact

of UVB has a clear effect on TEWL and instances of localized penetration, the

loosening of intercellular structures may be countered by the increase in epider-

mal thickness and the increased number of stratum corneum layers, which would

increase the observed lag time. However, the model observed in our data provides

hints at a mechanism that is challenging to observe in thin histological sections

and may be responsible in some part for the disparities that exist in the literature

with QDs [33] and other NPs. [34–36]


B

D

A

C

Brightfield QD Fluorescence QD Enhanced LUT

Figure 3.3: Microscopy of Histological SectionsCollection and penetration trends of QDs with non-UVB irradiated mice (A) and(B) and those receiving 360 mJ/cm2 UVB 3.5 days before QD application (C) and(D). Brightfield, QD fluorescence, and a QD fluorescence look up table (LUT) tohighlight QD locations are shown for all conditions with equal fluorescence inte-gration times. Follicular accumulation of QDs is observable in (B) and (D), withUVB exposure increasing ability of QDs to passively diffuse through the barrier.A clearly observable hyperproliferation response is present when comparing (A)and (C), and there is a notable increase in skin penetration of DHLA coatedCdSe/ZnS QDs with UVB exposure. However, the penetration levels after UVB(C) are an extreme example, of which few locations exhibiting such a large amountof diffusion through the stratum corneum barrier were found. Scale bar is 10 µm.


3.3.4 Ultrastructural TEM Analysis of Penetration Path-

ways

When TEM with silver enhancement was used, the ability of QDs to penetrate

the skin was brought into even stronger contrast. The silver enhancement proce-

dure used was established in our previous work, [29] and serves to increase the size

and contrast of the QDs thereby allowing distinction of relatively low contrast

CdSe/ZnS QDs from the tissue background. For the no UVB case, collection of

QDs was primarily observed in the outer layers of the stratum corneum, with

some collection in the hair follicles (Figure 3.4). Some penetration through the

outer layers of the stratum corneum could be observed (Figure 3.4A), with QDs

appearing to move through the intercellular lamellar space as found in the previ-

ous chapter of this thesis. We were unable to find any instances of penetration of

QDs past the stratum corneum barrier, and used EDAX, an x-ray spectroscopy

technique, to ensure that any suspicious spots did not contain silver. For an event

that would be as scarce as a QD passing intact stratum corneum barrier, the

use of 70 nm thick TEM sections makes it extremely improbable that one will

be found. When considering the 360 mJ/cm2 UVB samples, some distinct mor-

phological and QD penetration differences become apparent (Figure 3.5). The

stratum corneum of all samples is substantially thickened and the intercellular

connections appear much looser. Nuclei and unprocessed lamellar granules are

visible in a number of locations at the outer portions of the stratum corneum,

which is a common feature of UVB damage and may contribute to barrier impair-

ment. Additionally, the number of cell layers between the bottom of the stratum

corneum and the dermal/epidermal junction is is higher with an overall increase

in thickness. The differentiating keratinocytes throughout the epidermis appear


to have looser junctions as well, and their shape and morphological marker for

the stages of differentiation are impaired. The changes observed translated to

higher skin penetration levels, as the 360 mJ/cm2 UVB samples did have sub-

stantial QD stratum corneum penetration (Figure 3.5). In every slice, the silver

enhanced QDs were able to make it further through the loosened outer layers of

the stratum corneum in the intercellular space (Figure 3.5A) as expected, and

some occurrences of corneocytes filled with large amounts of QDs were observed

(Figure 3.5A,i), suggesting that isolated areas with relatively higher amounts of

stratum corneum damage may allow less hindered diffusion of QDs through the

stratum corneum. Similarly to the fluorescence histological imaging, scarce but

high penetration levels through the stratum corneum were observable (Figure 3.5).

These locations were not found in the non-irradiated samples and silver presence

was confirmed using EDAX. To find support for the trend, a large number of slices

had to be examined, which further supports the idea of high penetration localized

areas as found during histology, and emphasizes the need for examination of many

sections and the inability to accurately quantify the rate of skin penetration for

substances based on TEM or fluorescence histological analysis. The small sam-

pling area achievable is a strongly limiting factor. However, the non-QD control

samples each have very low levels of silver, which suggests that the noise floor for

TEM is very low (Figure B.4, Appendix B).

3.3.5 Quantitative Distal Organ Analysis

Histological and TEM analysis provide important insight into localized skin pen-

etration patterns and some understanding of penetration mechanism, but do not

yield quantitative results. To evaluate the impact of UVB on skin penetration of


B

A

i.

i.

i.

ii.

ii.

i.

Figure 3.4: Control TEMWithout UVB irradiation, the collection of QDs is confined mostly to the upperlayers of the stratum corneum (A) as observed by TEM. With close examination,the silver enhanced QD morphology can be observed and confirmed by EDAXspectroscopy (i). When lower portions of the stratum corneum and the rest of theepidermis are examined, no evidence of silver enhancement can be observed (B)in the more superficial stratum granulosum (i) or in the stratum basale (ii).


Organ Radiation Dose Vehicle QD

Organ Mass (g) Cd Mass (ng) Organ Mass (g) Cd Mass (ng)

Liver 0 mJ/cm2 UVB 1.78±0.26 3.68±1.24 1.78±0.27 3.76±0.26

360 mJ/cm2 UVB 1.71±0.36 5.30±1.16 1.65±0.25 8.21±2.27

Lymph 0 mJ/cm2 UVB 0.03±0.009 N.Q. 0.02±0.006 1.30±0.62

360 mJ/cm2 UVB 0.02±0.003 N.Q. 0.02±0.006 0.57±0.25

Table 3.1: Cd Organ AccumulationOrgan mass and Cd accumulation for vehicle-only and QD-applied mice (n=4).Consistent organ harvesting and Cd presence can be observed for each treatmentcondition. For non-irradiated and irradiated vehicle-only samples, Cd lymph nodeaccumulation is below the limit of quantification (N.Q.).

QDs at a systemic level, it is necessary to look at organ translocation patterns.

The total dose we applied was estimated to be 105.0 picomoles of QDs, which

was evaluated using atomic absorption spectroscopy to equal 112.7±2.4 µg Cd.

The total organ and Cd mass accumulation yielded interesting trends (Table 3.1),

and were transformed to a ng/g measure for clarity of graphical representation

(Figure 3.6). 2-way ANOVA analysis of the total Cd in the liver found that UVB

strongly impacted Cd accumulation (p<0.005) and that QD application was a

near-significant effector (p=0.058). This result is due to the lack of QD accumu-

lation in the liver without UVB exposure. 1-way ANOVA analysis of the lymph

nodes found a significant difference in Cd accumulation due to UVB (p<0.05).

When examined in more detail, analysis of the non-irradiated and UVB mice

without QD exposure found statistically indistinguishable levels of Cd in the liver,

and Cd accumulation in the regional lymph nodes below the limit of quantification

in all vehicle control samples. With QD exposure to the non-irradiated mice, the

Cd levels in the liver were 2.15±0.56 ng/g, with no statistical difference from

the control (p=0.45). However, an unexpected increase in the Cd accumulation

in lymph nodes could be observed, with values reaching 64.1±22.6 ng/g, which

equates to 0.0012% of the applied dose (Figure 3.6). All lymph node samples from


vehicle exposed (no QDs) animals had Cd content below the limit of quantification,

and all lymph node samples from QD exposed animals had Cd content above the

limit of quantification. The experimental care that was taken in combination

with a low observed standard deviation percentage (35% of the mean versus 49%

of the mean for the no QD sample) suggest that observation of Cd accumulation

in the lymph nodes for non-irradiated animals is not an artifact, such as might

occur with QD contamination of dissection implements. To confirm these findings,

an additional control was performed with QD application to intact barrier and

sacrifice at a 0 h time point. This control found no Cd accumulation in the

liver and lymph Cd below the limit of quantification. When irradiated with 360

mJ/cm2 UVB, liver Cd presence increased from 3.29±1.33 ng/g in the control

to 5.05±1.51 ng/g. When compared to the UVB vehicle control, the difference

between these values reached statistical significance using a Students unpaired

t-test (p<0.05). This liver value equates to 0.0073% of the applied dose. Lymph

node samples for the 360 mJ/cm2 UVB irradiated QD mice were elevated as well

to 25.1±14.6 ng/g or 0.0005% of the applied dose. Interestingly, the value was

lower than that for the non-irradiated QD mice (p<0.05). This result can be

explained by changes in UV-induced immune status as described below.

3.4 Discussion

The skin has had a great amount of research effort focused on determining the

ability of NPs to penetrate intact and damaged barrier. The results in this chap-

ter find low levels of skin penetration for intact murine skin, with an increased

but still low level maintained in UVB disrupted skin barrier. Importantly, this

is some of the first research to apply quantitative techniques to the evaluation of


C

B

Ai.

i.

i.

i.

ii.

ii.

i.

i.

ii.

ii.

Figure 3.5: QD Skin Penetration by TEMExposure to 360 mJ/cm2 UVB increases the skin penetration of QDs in a mouse.QD collection in (A,i) and penetration through and between the UVB-damagedouter corneocytes (A) is commonly found. Instances of silver enhanced QDs dif-fusing through weaknesses in the stratum corneum and epidermis are found in thestratum granulosum (B) with a tendency to move in the cellular boundaries (i, ii).Similar observations can be made in the stratum basale region (C), where long-lasting and highly proliferative basal keratinocytes reside. EDAX confirmation isused to ensure that the particles are silver enhanced QDs.


QD skin penetration and so expected results are not yet clearly defined. That

being said, our results in intact skin do match up well to the existing literature

for QDs. Results reported in the previous chapter have found very low to un-

detectable amounts of QD skin penetration for intact barrier with carboxylated

round QDs of 15-20 nm hydrodynamic diameter in murine skin, and another study

from our lab has found undetectable QD penetration into the epidermis with the

same DHLA coated QDs used in this study in ex vivo human skin. [5] Expanding

on their earlier research that found skin penetration through porcine skin for posi-

tive, neutral, and negatively charged QDs, [33] recent work by the Monteiro-Riviere

group has demonstrated minimal penetration in intact skin ex vivo porcine skin

using neutrally charged PEG-ylated nail-shaped QD with a 40 nm hydrodynamic

diameter [3] and ex vivo rat skin using negatively charged carboxylic acid coated

round and ellipsoidal QDs with 14 nm and 18 nm hydrodynamic diameter, re-

spectively. [4] Gopee et al. had similar findings with in vivo SKH-1 hairless mouse

skin using PEG-ylated nail-shaped QDs with a 40 nm hydrodynamic diameter

and perhaps the first reported quantitative evaluation, with organ analysis of the

liver and lymph nodes, as well as supporting fluorescence microscopy. [6] Our lower

standard deviations and basal Cd levels observed may contribute to the differ-

ences between our study and that of Gopee et al. Additionally, their usage of

nail-shaped QDs with a larger hydrodynamic radius and PEG surface coating

may alter the ability of lymphatic cells to transport the QDs to the lymph nodes.

PEG surface coating is well accepted to alter the collection and uptake of NPs in

the body, which could decrease their observed signal in lymph nodes. [37] Another

factor may be our usage of SKH-1 mice that have been backcrossed with the C57

strain, which may provide a more robust immune response than the inbred SKH-1

mouse strain. It is important to note that our values for the lymph node accumu-


lation are very low proportions of the overall applied dose. In another important

quantitative study, a recent article by Lopez et al. found that QDs with posi-

tive, negative, and neutral surface coatings and hydrodynamic diameters ranging

from 10-22 nm were able to penetrate split thickness ex vivo porcine skin in low

amounts. [7] The amounts quantified in the dermis by physical separation of the

dermis and epidermis were quite low but seemingly significant (0.078% for the

highest penetrating QDs whose core size and emission peak were not mentioned)

and the levels of Cd observed in skin without QD treatment was not reported.

These reported results match up well to those found in this chapter, where we see

just 0.0012% of the applied dose to intact skin being collected in the lymph nodes.

Providing additional support for our findings is research by Vogt et al. that has

demonstrated the uptake of polymer NPs with hydrodynamic diameter of 40 nm

by Langerhans cells, the major epidermal dendritic cell type. [38] These cells are

trafficked to the lymph nodes, and though the measured total amount of QDs

in the lymph nodes at 24 hours is quite low, presence suggests the possibility of

an active QD uptake process by antigen presenting cells. [39,40] If skin penetration

by other types of metal-based NPs is considered, similar results have been found

in maghemite and iron NPs with a particle size of 5 nm and 50 nm, which were

determined to minimally penetrate ex vivo human skin, albeit to detectable levels,

in a static diffusion cell. [41,42] Similarly, NP-sized TiO2 and ZnO with hydropho-

bic coatings, [43–48] hydrophilic coatings, [44,49] and no coating [36,49,50] demonstrated

minimal to undetectable skin penetration in several skin models.

However, with barrier disruption such levels of skin penetration have the pos-

sibility of being increased. In our work, the clinically relevant barrier disruption of

UVB exposure is used, and is able to increase the skin penetration of QDs. This

dissertation finds UVB induced barrier disruption as measured by TEWL that


1.0

10.0

100.0

Liver Lymph

[Cd]

(ng/

g)

0 mJ/cm2 UVB No QD0 mJ/cm2 UVB QD

360 mJ/cm2 UVB No QD360 mJ/cm2 UVB QD

N.Q. N.Q.

Figure 3.6: Elemental Organ AnalysisAtomic absorption spectroscopy of the distal collecting organs to determine tissueCd concentrations. Statistical significance between groups is marked by lines (1line, p<0.05). Without UVB exposure, there is no evidence of Cd accumulationin the liver. After UVB exposure, QD exposure increases Cd levels in the liver(p<0.05). In the lymph nodes, vehicle control treated animals exhibit Cd belowthe limit of quantification. Without UVB, a significant presence of Cd can beobserved in the lymph nodes. With UVB, significant Cd levels are again foundin the lymph nodes. For QD treated lymph nodes, Cd level decreases after UVBexposure (p<0.05).


matches agrees well with previous literature SKH-1 mouse model, [11,12,15,16,20,21]

and is thought to be due to a combination of proliferation and immunologic re-

sponses to UVB that alter the epidermal calcium gradient [20] and status of lipids

in the stratum corneum. [16] With regard to the permeation of applied agents,

the only other study that we are aware of in the literature is a 1984 study by

Lamaud and Schalla, wherein they investigated the effects of UVB on TEWL of

hairless rats at a range of doses from 165-1320 mJ/cm2 UVB, and applied 14C-

labeled hydrocortisone to the 660 mJ/cm2 UVB dosed rats for various time points

up to 24 hours over the TEWL peak. [19] They were able to find clear evidence

of an increase in the rat skin penetration that corresponded well to the TEWL

observed in their studies. Their findings support a matching outside-in barrier

disruption that is observed by us using liver elemental analysis and microscopy

with QD application. For skin penetration of NPs, the only other study that we

are aware of is the the work reported in the previous chapter that investigated

the ability of carboxy-coated 565 nm emitting QDs with hydrodynamic diameter

of 20 nm to penetrate intact and 270 mJ/cm2 UVB irradiated murine skin imme-

diately after UVB exposure. Despite similarities in collection patterns between

non-irradiated and UVB irradiated mice, we observed very rare occurrences of

QD in the epidermis of healthy tissue, and a higher occurrence- albeit still quite

low- of QDs penetrating skin that was exposed to UVB. Our quantitative work

described herein examines QD skin penetration over the peak TEWL time period,

and is able to find an ability for QDs to penetrate the disrupted stratum corneum

barrier and possibly collect systemically in the liver at low levels (0.0073% of the

applied dose) with a 360 mJ/cm2 UVB dose.

Skin penetration of QDs after barrier disruption has been found for other bar-

rier disruption techniques as well. An early study used low frequency sonophoresis


to impair the skin barrier, a common drug delivery enhancement technique, and

found a substantial increase in QD skin penetration (coating and hydrodynamic

size not stated) in an ex vivo porcine skin model. [31] Recent work has followed

up on that research using a synergistic combination of sodium lauryl sulfate and

sonophoresis to increase the penetration of QDs with positive, negative, and neu-

tral charges and hydrodynamic diameters ranging from 10-20 nm as well as 4.3 nm

negatively charged gold NPs in full thickness and dermatomed ex vivo porcine skin

using a physical separation technique and mass spectroscopy. [7,51] Research in our

lab has found low levels of negatively charged QD skin penetration with hydrody-

namic diameter of 14 nm through healthy ex vivo human skin with an increase in

stratum corneum penetration when ex vivo human skin is treated with a depila-

tory agent or tape stripped using flow cytometry. [5] Interestingly, independently

conducted research using nail-shaped PEG-ylated QDs with hydrodynamic diam-

eter of 40 nm by Gopee et al. on in vivo mice and Zhang et al. on ex vivo rat skin

has found that tape stripping, a well-accepted method of barrier disruption [52] and

permeability enhancement of large hydrophilic molecules, [18] has no impact on QD

skin penetration. [4,6] The difference in results could be indicative of a difference in

tape stripping technique, and highlights the importance of establishing alternative

techniques for the evaluation of local epidermal skin penetration. When the more

aggressive barrier disrupting technique of dermal abrasion was used, both groups

were able to observe a substantial increase in the skin penetration of QDs. Der-

mal abrasion is an invasive technique that eliminates the stratum corneum and

much of the epidermis completely to allow free access of QD to the dermis and

vasculature. With other metallic NPs, dermal abrasion or chemical penetration

enhancer application have been shown to increase the skin penetration of silver

NPs [34] and ZnO NPs, respectively. [32] Even though some differences exist in the


detailed levels of impairment that are necessary to effect the skin permeability

to QDs or other metallic NPs that may be due to particle size/shape, particle

coating, or skin type, it is clear that disrupting the skin barrier can increase risk

of NP penetration, a conclusion that this work supports.

As the research presented herein discusses, UVB increases skin penetration

to levels that can be observed to collect systemically in the liver and the lymph

nodes at low levels. The research discussed above by Gopee et al. found that their

PEG-ylated QDs applied to dermabraded mouse skin collected most definitively in

the liver and the regional draining lymph nodes (axillary and brachial), and so we

focused our analysis on these organs. [6,53] The decreased lymph node accumulation

observed over control mice can be attributed to the loss of epidermal Langerhans

cells, a process that occurs soon after exposure and lasts for 4-14 days. [54–56] Since

we observe significant levels of Cd in the lymph nodes of non-irradiated animals

with no evidence of substantial penetration using other techniques, and evidence of

increased penetration with microscopy techniques with significant levels of Cd liver

accumulation with UVB irradiated mice, we conclude that liver Cd accumulation

has greater potential as a measurement of systemic-level penetration following

24 hr QD exposure. In our experiments, the statistically significant but still

low skin penetration of QDs found with TEWL increase suggest that the barrier

disruption induced by UVB does not provide enough of a disruption to enable

a large magnitude change in quantitative liver Cd accumulation at 24 h. When

the fold change over control of TEWL induced by UVB is compared to physical

barrier disruption, the level reached in this chapter has been shown to be sufficient

to cause a substantial increase in penetration PEGmolecules with a size of 1 kD. [18]

Importantly, the change in PEG molecule penetration relative to TEWL is altered

by the mechanism of barrier defect and so an understanding must be established


for individual barrier disruption mechanisms. The barrier disruption caused by

UVB in these experiments evidences an increase in QD skin penetration ability,

but one whose systemic collection is low over the conditions studied. Doses of UVB

that would impact barrier strongly enough to induce full penetration of QDs and

allow fully predictive evaluation of barrier function is therefore expected to be on

a necrotic UVB dose level. The loosening of stratum corneum cellular junctions

and disorganization of the intercellular lipid lamellae are counteracted by the size

of the pores and increase diffusion distance before the stratum corneum barrier

is cleared by the QDs, and so the loosening must achieve high levels to overcome

the increased tortuosity and path length. However, with repeated UVB doses or

repeated QD application doses higher QD penetration levels may be observable,

a question that future work could address. Our results presented in this chapter

demonstrate that QDs are transported to the regional draining lymph nodes at

low levels in healthy skin, and that this response is decreased by UVB exposure.

We have also found that UVB irradiation disrupts the skin barrier and increases

the ability of QDs to penetrate the stratum corneum in isolated instances that

reach detectable levels in the liver. Importantly, all of these events are at very

low proportions of the applied dose. The ability of QDs to penetrate the skin at

low but quantifiable levels is an important discovery that motivates future work

to generate a mechanistic understanding at the cellular level of the effects of UVB

dose and QD application time as well as the impact of NP size, surface coating,

shape, and core chemistry on skin penetration. This work will enable the design

of minimally penetrating cosmetics or use UVB to assist with drug delivery.


3.5 Conclusions

In conclusion, this portion of this thesis has demonstrated that UVB irradiation

causes a strong impact on skin barrier function that can be quantitatively related

to the skin penetration of QDs. We have also found the first instance of topically

applied QD trafficking to the lymph nodes in healthy animals. This trafficking

is decreased by exposure to UVB, presumably from the well-accepted impact of

UVB on Langerhans cell presence, the details of which can be explored in future

work. We have determined microscopically that there is a clear UVB induced

increase in the penetration of QDs that achieves a borderline significant level in

the liver, but still remains at a low level of the applied dose. Difficulty in finding

occurrences of penetration using histological sectioning techniques despite quanti-

tative differences suggest a “seed point” penetration model through low frequency

pores as introduce in previous literature. Results suggest for the first time that

QDs can be trafficked to the lymph nodes in healthy skin and UVB irradiated

skin at low levels, and increase awareness about the possibility of UVB impact

on NP skin permeation. As quantification techniques improve, it grows increas-

ingly important to determine the impact skin barrier function and NP size, shape,

surface chemistry, and mechanical properties on skin penetration potential to es-

tablish accurate risk assessments for NPs. In the case of UVB barrier disruption,

a condition that is likely to occur with TiO2 and ZnO NP exposure, further re-

search that integrates the NPs commonly used in sunscreens will help to assess

potential risk factors. Future studies are called for that will continue to refine the

quantification of barrier disruption and NP penetration to address these concerns

and help provide regulatory guidelines for NP exposure and implementation.


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137

Chapter 4

UVB, QDs, and Primary

Keratinocytes

The impact of UVB and differentiation stateon QD interactions with primary keratinocytes 1

1Portions of this chapter are adapted from Mortensen, L.J., Ravichandran, S., and DeLouise,L.A. The impact of UVB and differentiation state on quantum dot interactions with primarykeratinocytes, submitted to Nanotoxicology 2011.

CHAPTER 4. UVB, QDS, AND PRIMARY KERATINOCYTES 138

4.1 Introduction

Previous sections of this dissertation have established the ability of UVB to in-

crease skin penetration of QDs and investigated the distal organ collection that

results. Areas that have yet to be investigated in this thesis are the interaction

of QDs with the local epidermal cellular environment that would result from QD

skin permeation and the effects of UVB exposure on this process, which will be

the focus of this chapter.

As discussed in previous sections, research by a number of groups has found

that QD skin penetration is increased by barrier compromise such as low fre-

quency sonophoresis, [1] tape stripping, [2] dermal abrasion, [3,4] and the UVB expo-

sure investigated in this thesis. These studies suggest that depending on the QD

characteristics and the severity of the insults, barrier compromised skin can be

breached by QDs, which are then able to collect systemically [5] and interact with

the local cellular environment in a potentially toxic fashion. [6–9]

Skin exposure to UVB has been demonstrated in this thesis and in the lit-

erature to cause a change in barrier function that peaks several days after an

acute exposure. [10,11] This thesis has determined that this barrier defect can be

related to an increased risk of qualitative skin penetration of QDs immediately

after exposure and quantitative skin penetration of QDs 3.5- 4.5 days later. These

results are important, as UVB damage to the skin barrier is commonly combined

with the application of cosmetics such as sunscreens, such as could occur with

inadequate application or overexposure to the sun. An increased penetration of

QDs after UVB exposure suggests the need for understanding the effects of UVB

on interaction of QDs with the predominant skin cell type, the keratinocyte, as a

requisite next step. UVB has a strong cellular effect on keratinocytes in the skin,


with the flood of cytokines and prostaglandins generated after UVB exposure in-

ducing DNA synthesis as a marker of the strong proliferative response. [10,12–16]

This proliferative response and an additional immune-regulated component have

been suggested to induce the barrier disruption response, [10] which is correlated

to a loss of the epidermal calcium gradient [11] and a disorganization of stratum

corneum lipids. [17] Additionally, there is activation of keratinocyte growth factor

receptor (KGFR) and the PAR-2 pathway, which has the effect of increasing ker-

atinocyte phagocytosis of melanosomes from neighboring melanocytes in response

to UV exposure. [18–20] Despite the current controversies about the mechanism of

melanin transfer to keratinocytes, [21] a distinct increase in melanin production in

the melanocytes and transfer to keratinocytes that occurs after UVB exposure is

clear. [22] Although these profound differences in cellular activity that are induced

by UVB are well studied, very little literature examines the impact of UV radi-

ation on NP interaction. Several studies have found increased cellular reactive

oxygen species generation and toxicity of TiO2 NPs with UVA exposure. [23–28]

With QDs, one seminal study has determined that there is a substantial increase

in the toxicity of carboxylic acid coated CdTe QDs when cells are pre-exposed

to QDs and subsequently irradiated with UVA light. [29] The application of their

results to our study is limited, as they used UVA light, large doses of QDs, pan-

creatic cells, and irradiated the cells with the QDs already present. The question

of UV influence on toxicity of NPs already present in cells is important, but for

our studies we wish to decouple the response of QD photoexcitation that UV ra-

diation engenders, [30] and focus on the biological response to the more bioactive

wavelengths of UVB as it pertains to QD presence.

It is important to note that most of the UVB-induced responses mentioned af-

fect the proliferative keratinocytes, which are cells along the basement membrane


of the epidermis. The epidermis of the skin can be divided into these proliferative

keratinocytes, which divide continually to supply the epidermis; and differenti-

ated keratinocytes, which are separated in layers commonly defined by cellular

morphology (i.e. stratum spinosum, stratum granulosum) and undergo termi-

nal processes to complete transformation into the corneocytes and intercellular

lipid lamellae that make up the stratum corneum barrier. Additionally, other

changes such as the expression and activity of EGFR and low density lipopro-

tein (LDL) receptor, commonly recognized to be important in the uptake of

melanin and other endocytic processes are more active in the proliferative basal

keratinocytes, particularly following UVB exposure. [31–35] In terms of QD inter-

action, only proliferative-type keratinocytes have been investigated, with toxicity

and uptake mechanisms of a variety of commercially available QDs examined by

the Monteiro-Riviere group. Their studies have suggested toxicity limits consis-

tent with the literature for other cell types for QDs with positive, negative, and

neutral surface charges (approx. 20 nM QDs for all surface chemistries). [36–38] Ad-

ditionally, their recent work has suggested a putative keratinocyte uptake mecha-

nism for negatively charged QDs that is governed predominately by proliferative

keratinocyte LDL receptors, [39] among others. However, they did not address

any differences that may arise between basal proliferative and differentiated ker-

atinocytes in the skin. If basal keratinocytes endocytose QDs, there is an increased

risk of cell death, dysfunction, or transformation, as these cells are anchored to

the basement membrane and produce daughter cells for long periods of time in the

epidermis. [40] This chapter eliminates the ROS generative and cytotoxic effects of

UV light on QD and examines the biological impact of UVB on cellular interaction

with QDs by exposing cells to UVB first and then adding QDs. This work expands

on the current literature by examining the way that the cellular UVB response


changes the way that primary proliferative and differentiated keratinocytes inter-

act with QDs using flow cytometry to quantify cellular uptake. Additionally, we

examine for the first time potential differences in QD interaction for proliferative

versus differentiated keratinocytes, providing critical insight needed for assessing

risk from QD skin exposure.


4.2.1 QD Functionalization

We functionalized 620 nm emitting CdSe/ZnS QDs purchased in toluene at a

concentration of ∼10 µM (NN-Labs LLC) with dihydrolipoic acid (DHLA) for

use in an aqueous environment as described in the previous chapter. Briefly, a

250 µL aliquot of organic QDs are removed from their solvent by addition of 1.5

mL of a 50/50 methanol/acetone mixture and centrifugation at 12,000 rpm. The

excess solvent is evaporated using nitrogen gas and the QD resuspended in 250

µL tetrahydrofuran (THF). Meanwhile, a 10,000x molar excess of pure DHLA (50

µL) is added to 1 mL methanol in a different glass vial and the pH adjusted using

tetramethylammonium hydroxide pentahydrate powder (Sigma-Aldrich Inc.) to

pH=11.0. The QDs in THF are added dropwise to the methanol mixture and

incubated at 60C with stirring for 3 hours. After three hours, the temperature

is reduced to room temperature and the reaction mixture is stirred overnight.

The bottom methanol layer is then removed, shaken with excess ether, and cen-

trifuged at 12,000 rpm for 10 minutes. The liquid is decanted, the pellet dried

using nitrogen gas, and the QDs resuspended in 250 µL double distilled water. To

remove any excess ligand, the functionalized QDs are dialyzed using a 5kD molec-


ular weight cutoff DispoDialyzer filter (Harvard Apparatus Inc.) and an excess

volume of water for 72 hours. After dialyzing, the concentration is determined by

measuring the absorption at the first exciton and using an extinction coefficient

from the literature with Lambert-Beer’s law. [41]

4.2.2 Primary Keratinocyte Isolation, Culture, and Differ-

entiation

Fresh viable human skin from healthy adult donors was obtained following ab-

dominoplasty or mammoplasty (Strong and Highland Hospitals, University of

Rochester, NY), stored at 4C, and used within 6 hr of surgery. Skin samples

were approved for usage by the University of Rochester Research Subjects Re-

view Board. To harvest basal keratinocytes, a modified version of the protocol

outlined by Pentland and Needleman was used. [42] First, skin samples were rinsed

with sterile 1x phosphate buffered saline (PBS), treated with 0.4 mL fungizone

(Invitrogen) in 500 mL sterile 1x PBS for 10 min, and rinsed again thoroughly

with 1x PBS. Subcutaneous fat was removed and the dermis thinned using a ster-

ile blade and scalpel. The skin samples were then transferred to fresh 100 mm

sterile tissue culture plates with gauze and incubated overnight at room temper-

ature in 12 mL of 0.25% Trypsin-EDTA in a sterile cell culture hood with the

stratum corneum exposed to the air. The epidermis was carefully separated from

the dermis and mixed well with serum free keratinocyte growth media (KGM-SF)

and 1% penicillin/streptomycin (Gibco Inc.) plus 10% fetal bovine serum (FBS).

After mixing, the cells were strained using a 100 µm filter and plated in collagen

coated 12 well plates (Purecol 1:5 diluted in 1x PBS). After 24 hours, the media

was changed to KGM-SF without FBS, and three days later differential trypsin


(∼35-45 sec incubation with 0.25% Trypsin-EDTA) performed to remove resid-

ual melanocytes and fibroblasts while leaving behind the keratinocytes. After 1

week, the media was changed again. When the plates reached 80% confluence (∼2

weeks), the cells were split into two groups, one with low calcium (0.09 mM) and

one with high calcium (1.5 mM Calcium Chloride) to induce differentiation.

4.2.3 UVB Irradiation

Experiments were performed 48 hours after the addition of calcium to half of the

harvested cells. The media was removed from both proliferative and differenti-

ated cells and replaced with 300 µL 1x PBS. Cells were then exposed to through

a Schott WG 295 nm long-pass glass filter (BES Optics) using FS20 sunlamps

(Westinghouse) as described previously by Brouxhon et al. [43] The FS20 sunlamp

emits primarily in the UVB spectrum (290-320 nm), with low amounts of UVA.

Lamp output was calibrated using an IL1700 light meter (International Light)

with an SED 240 probe to detect light output from 255-320 nm (wavelengths

shorter than 295 nm removed by Schott filter). Doses from 20-140 mJ/cm2 UVB

were established using this technique. To determine the impact of UVB dose on

cellular viability, 3 wells of each calcium state were exposed to a UVB dose rang-

ing from 0-140 mJ/cm2 and mitochondrial reductase activity measured 24 hours

later by the MTT assay (Invitrogen). Briefly, 100 µL yellow MTT reagent (1.2

mg/mL) was added to each well and incubated for 4 hours. The reactive solution

was then removed, replaced with 1 mL isopropanol to solubilize the resultant for-

mazan crystals, and the absorbance at 600 nm measured. UVB dose impact on

viability using MTT was measured on 4 separate occasions with cells grown from

unique donors and viability was statistically compared to unexposed control using


Student’s paired t test.

4.2.4 QD Application

To determine the impact of exposure to increasing doses of DHLA QD 620nm

on cell viability, proliferative and differentiated primary keratinocytes were incu-

bated with QD for 24 hours with and without UVB exposure. Half of the cells

were exposed to 40 mJ/cm2 UVB, a dose that did not demonstrate statistically

significant killing of the primary keratinocytes, but that has been shown by pre-

vious work to induce biological response. [43,44] Doses of 0 nM, 0.5 nM, 5.0 nM,

and 50.0 nM DHLA QD 620 nm were then introduced into the cell media and

incubated for 24 hours in triplicate. The effect of QD on mitochondrial reductase

activity was measured as described above using the MTT assay on 4 separate

occasions with cells grown from 4 different skin donors and viability was statisti-

cally compared to unexposed control using Student’s paired t test. QD interaction

with cells was imaged using phase contrast and fluorescence microscopy (Olympus

IX70 with QImaging Retiga EXi camera) with a mercury lamp excitation source

(360/30 bandpass filter) and narrow emission (620/10 bandpass filter). Images

were analyzed using ImageJ. Parallel experiments were performed using the same

QD exposure protocols, but the flow cytometric preparation described below.

4.2.5 Flow Cytometric Analysis

To prepare the UVB irradiated and QD exposed cell samples for flow cytometric

analysis, the media was collected from each cell sample, pooled together with each

of their three repeats, and the cells were trypsinized from their plates. After ad-

dition of FBS, the cells from each condition were added to their respective media


and centrifuged. This procedure was enacted to preserve any cells that may have

been released from the plate into the media due to cytotoxicity over the course

of the experiment. The cell samples were then washed with 1x PBS, centrifuged,

and stained using a Calcein AM live stain (Invitrogen, Oregon, USA) and Sytox

Blue dead stain (Invitrogen, Oregon, USA). Each cell sample was incubated for

10 min in the dark in a 600 µL mixture of the two stains, both of which were

at 1 µM concentration. The samples were then centrifuged and the resuspended

in 3% formalin (VWR International) for analysis. The appropriate single stain

compensation controls were prepared for Sytox, Calcein, and QD using HaCaT

keratinocyte cells. The samples were analyzed using an 18-color BD LSRII flow

cytometer with filters for Calcein AM (488 nm ex. 515/20 nm em.) Sytox Blue

(405 nm ex. 450/50 nm em.) and QD (405 nm ex. 660/40 nm em.), and results

processed using Flow Jo (Version 7.6) software. We gated the forward scatter-

ing (FSC) and side scattering (SSC) plot to eliminate debris and multi-cellular

events, and the Calcein/Sytox plot to allow separation of live and dead cellular

fractions. The QD median intensity values prepared from 4 different skin donors

were averaged and compared using the Students t-test.

4.3 Results

4.3.1 Cellular Toxicity of UVB

When examining cellular response to UVB and QDs, the first concern is under-

standing the cytotoxic effect of UVB in the absence of QDs. To this end, we ex-

posed cells to increasing doses of UVB and determined cell viability after 24 hours

using MTT (Figure 4.1). Cell viability for both proliferative and differentiated


0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100 120 140

Rel

ativ

e Vi

abilit

y (M

TT)

Dose UVB (mJ/cm2)

low calciumhi calcium

* * **

*

** **

**

*

****

*

**

* **

Figure 4.1: Primary Keratinocyte UVB CytotoxicitySummary of UVB dose impact on relative cell viability as measured by MTT.UVB causes a statistically significant detriment to cell viability starting at 60mJ/cm2 for the proliferative keratinocytes versus their 0 mJ/cm2 dose. For

differentiated keratinocytes, at all UVB doses there is a statistically significantdifference versus the proliferative keratinocyte 0 mJ/cm2 dose (against which

they were normalized). However, at no dose did UVB prove toxic fordifferentiated keratinocytes versus their own 0 mJ/cm2 dose control. Sampleswere compared to the proliferative keratinocyte 0 mJ/cm2control using the

Students t test with ∗ = p < 0.05 and ∗∗ = p < 0.01.


primary keratinocytes is plotted normalized to the 0 mJ/cm2 UVB proliferative

control. Results showed an onset of toxicity (p<0.05) at 60 mJ/cm2 by MTT for

the low calcium proliferative keratinocytes. When cells were differentiated by the

addition of calcium, no statistically significant relative toxicity onset (p<0.05) was

observable versus the 0 mJ/cm2 differentiated cells at any dose. However, when

compared to the unexposed proliferative cells a statistically significant difference

(p<0.05) was observable at all UVB doses. When statistical comparison was made

between differentiated and proliferative cells at each individual UVB dose, a dif-

ference (p<0.05) was only seen at 0 mJ/cm2, 20 mJ/cm2, and 40 mJ/cm2 UVB

(none of which were toxic to the proliferative cells), suggesting that differentiated

cells have a lower basal metabolic activity than proliferative, but demonstrate the

same toxicity levels as the proliferative keratinocytes beginning at 60 mJ/cm2. To

maximize the difference between proliferative and differentiated keratinocytes, 40

mJ/cm2 was chosen for subsequent experiments.

4.3.2 Cellular Toxicity of QDs

To determine the cytotoxic effects of QDs on proliferative and differentiated ker-

atinocytes with and without UVB, we used a similar strategy as described above.

MTT was performed on proliferative and differentiated keratinocytes with and

without UVB exposure at a range of QD doses (Figure 4.2). For the low calcium

proliferative cells (Figure 4.2A), QD exposure of 50.0 nM induced a statistically

significant level of cytotoxicity (p<0.01) with and without UVB exposure. The

high calcium differentiated cells (Figure 4.2B) also demonstrated definitive toxic-

ity at 50.0 nM (p<0.05) using the MTT assay with and without UVB. The lack

of any differences between proliferative and differentiated keratinocytes or UV ex-


posure suggest that differentiation state and UV exposure do not strongly impact

the susceptibility of cells to QD toxicity. The QD dose effect on viability was

also examined using a Trypan blue exclusion assay (data not shown), and similar

viability results were found. Due to its lack of toxicity in most samples, the 5.0

nM QD dose is used for subsequent experiments.

4.3.3 Fluorescence Microscopy

To visualize QD interaction with primary keratinocytes in the plate, we observed

the cells after 24 hours using fluorescence microscopy (Figure 4.3). As described

above, the cells were washed 2x using DPBS to eliminate any residual QDs that

may have settled out onto the cell surface or the plate. This treatment yielded

undetectable QD background on the plate for all conditions. In the prolifera-

tive keratinocytes without UVB, there appeared to be a large number of QDs

clustered in the perinuclear area, with punctate spots denoting the QDs (Fig-

ure 4.3A), with low non-specific cellular adsorption. When pre-exposed to UVB

light, the number of QDs present in the proliferative cells appears to increase a

significant amount (Figure 4.3B). However, the high calcium differentiated ker-

atinocytes displayed substantial amounts of non-specifically adsorbed QDs on the

cellular surfaces (Figure 4.3C and 4.3D) in addition to the appearance of QDs

that appeared to be internalized in the cells. The punctate QDs that appear to

be preferentially located inside the cellular cytoplasm consistently increased with

exposure to UVB (Figure 4.3D). We examined additional QD doses of 0.5 nM and

50.0 nM (data not shown), and were able to observe similar trends of difference

between proliferative and differentiated keratinocytes, and a strong increase in

QD presence with increasing dose and morphological evidence of toxicity at 50.0


0

0.2

0.4

0.6

0.8

1

1.2

0 nM 0.5 nM 5.0 nM 50.0 nM

Rel

ativ

e Vi

abilit

y

[QD]

low calcium no uvlow calcium uv

A

0

0.2

0.4

0.6

0.8

1

1.2

0 nM 0.5 nM 5.0 nM 50.0 nM

Rel

ativ

e Vi

abilit

y

[QD]

hi calcium no uvhi calcium uv

B

** **

** **

Figure 4.2: Primary Keratinocyte QD CytotoxicityDetermination of the cytotoxicity levels that result from increasing dosage ofDHLA functionalized QD 620 nm in proliferative low calcium media keratinocytes(A) and differentiated high calcium media keratinocytes (B). For both charts,MTT assay shows an onset of toxicity occurring at a QD dosage of 50.0 nM(∗∗ = p < 0.01).


nM QDs. Due to our rinsing protocol and the differences between proliferative

and differentiated keratinocytes, it is likely that the QDs are inside the cells as

suggested by previously published studies using confocal microscopy. [29,45,46] To

confirm our results and to provide a more precise quantification of QD cellular

association, we used flow cytometry in subsequent experiments.

4.3.4 Flow Cytometry and QD Uptake

To determine if cellular differentiation state had any impact on the cellular as-

sociation of QDs, flow cytometry techniques were used. We took a traditional

gating strategy to allow the separation of single cells from debris and multiple

cell clusters (Figure 4.4A) and used a Calcein AM/ Sytox Blue live/dead stain

combination to separate live cells from dead cells (Figure 4.4B). Experiments here

considered the live cell population primarily and found a clear increase in the

association of QDs with cells with increasing QD doses that can be observed in

overlayed histogram distributions (Figure 4.4C). Our gating strategies allowed the

consideration of the live and dead cell populations separately. We found similar

trends when comparing the two population groups, but used the live cell popu-

lation because of a consistently higher standard deviation of QD intensity within

the dead cell population. We suspect that this trend is due to the increased vari-

ation in cellular membrane permeability and metabolic activity within the dead

population, which includes cells at various stages of apoptosis.

The values reported are from the average of 4 experiments to provide an ac-

curate representation of the cellular uptake of QDs and limit the impact that a

specific cell donor may have on results. Since tissue donor sources for primary cells

are blinded, there is no possibility to control the age or skin type from which our


Low

Cal

cium

No

UV

Phase Contrast

Low

Cal

cium

UV

Hi C

alci

umN

o U

VH

i C

alci

umU

V

5.0 nM QD/DAPI overlayA

B

C

D

Figure 4.3: Fluorescence Microscopy of Cellular UptakePhase contrast and widefield fluorescent microscopy of the QD-cell interaction.Each treatment condition was either irradiated with 40 mJ/cm2 UVB or shamirradiated and incubated with 5.0 nM QD 24 hours. The media was removed andreplaced with 1x DPBS to allow imaging in the plate. UV appears to increase theuptake of QD in (high calcium) differentiated primary keratinocytes (B) over thesham-irradiated samples (A). In proliferative primary keratinocytes (low calcium),UV also appears to increase the cellular uptake of QD (D) over sham irradiation(C). More importantly, the amount of non-specific adsorption in the differentiatedcells (A & B) is much higher than that in proliferative cells (C & D), despite apossible increase in internalized QD for the proliferative keratinocytes.


primary keratinocytes are harvested, which contributes strongly to cell behaviors

such as growth rate and morphology. When the median intensity values are aver-

aged over multiple experiments and plotted on a log scale, several trends become

apparent (Figure 4.5). Throughout, trends mirror the results of our fluorescence

microscopy experiments. The most noticeable trend is a dramatic increase in the

uptake or association of QDs with cells with increasing dose. There is a steady

increase of median fluorescent intensity with the 0.5 nM and 5.0 nM doses that

has a lower differential magnitude at 50.0 nM. The lower magnitude of increase

can be attributed to a saturation in the number of QDs that are able to associate

or be internalized by the cells and to nearing the saturation limit of the detec-

tor. Another strong trend is the impact of differentiation state on the uptake or

association of QDs with cells. A statistically significant difference between the

proliferative and differentiated cells was observed at the 0.5 nM and 5.0 nM QD

doses (Figure 4.5). This observation matches up well to observations made in QD

penetration of human tissue that suggest a preferential collection in basal ker-

atinocytes (unpublished results). When considering the impact of UVB, however,

the results are less clear. There is no statistically significant trend in differenti-

ated or proliferative cells at any dose, although the data at 0.5 nM and 5.0 nM

doses suggests UVB exposure may exacerbate uptake. Taken together, these re-

sults suggest that the dosing of QDs and differentiation state of cells has a strong

influence on the uptake or association with QDs and that UVB pre-exposure is

not a significant contributor, at least at the dosage and time point used in this

chapter.


50 nM QD5 nM QD0.5 nM QD0 nM QD

0 102 103 104 105

QD Fluorescence

0

20

40

60

80

100

Nor

mal

ized

Fre

quen

cy

0 50K 100K 150K 200K 250KForward Scatter

0

50K

100K

150K

200K

250K

Side

Sca

tter

64.6

Syto

x Bl

ue D

ead

Stai

n

61.734.2

0 102 103 104 105

Calcein AM Live Stain

0

102

103

104

105A B

C

Figure 4.4: Primary Keratinocyte Gating SchemeAn example cell experiment to demonstrate our flow cytometric gating strategyand effect on QD uptake. The forward scattering/side scattering plot (A) is gatedto eliminate debris and multi-cell clusters. The Calcein AM/ Sytox Blue live/deadscatter plot (B) is segregated into live and dead proportions to allow analysis ofthe more homogeneous live cell region. An example increase in QD fluorescencewith increasing dose of QD (low calcium without UV exposure) can be observedwith overlayed histograms (C).


4.4 Discussion

The research presented in this chapter has determined that the cellular differen-

tiation state of primary keratinocytes plays an important role in their interaction

with and uptake of QDs. Importantly, we are able to observe no distinct differ-

ence between cellular response to QDs with or without exposure to UVB. The

interaction between UVB and the skin has been an active area of research for

a number of years due to the well-accepted correlation between UVB exposure

and increased skin cancer rates. An additional concern in acute UVB dosing that

has been examined by several studies, including work presented in the preceding

sections of this dissertation, is a change in barrier function that peaks 3-4 days

after exposure. [10,11,17,47,48] In my disseration work, I have demonstrated that there

is a greater risk of QD skin penetration both immediately after UVB irradiation

and 3-4 days post UVB exposure. [48,49] This study has considered the interaction

of QDs with primary epidermal keratinocytes in a differentiated and proliferative

form, and inquired into the effects UVB irradiation may have on this process.

The relevant biological impacts of UVB on cellular events to our studies mostly

revolve around the proliferative response and cytotoxic effects of UVB. UVB ra-

diation exposure damages a number of cellular compartments, but the most im-

portant of these is well accepted to be DNA. In particular, the DNA absorption

of UVB light that reults in the formation of DNA pyrimidine dimers activates the

p53 pathway to induce DNA repair, proliferation, and apoptosis. [50,51] It has been

shown in recent studies that differentiated primary keratinocytes are more resis-

tant to UVB in terms of apoptosis induction, which results from an increase in

the Notch signaling pathway and may be related to an increased apoptotic resis-

tance to reactive oxygen species. [29,52,53] In the skin, it is more critical to limit the


maximum allowed damage from UVB on proliferative primary keratinocytes than

differentiated, as mutations in the proliferative cells can have more far-reaching

effects. Our results demonstrate a similar phenomenon with earlier induction of

UVB toxicity in the proliferative keratinocytes.

The contrast between differentiated and primary keratinocytes in terms of

stress response to UVB might suggest that there would be a difference in toxi-

city of QDs. However, the levels of toxicity measured by MTT for each of the

cell types reaches significance at the same QD dose, suggesting that nanoparticle

toxicity may be due to multiple pathways (cell association in the high calcium

differentiated cells and internalized QDs in the low calcium proliferative cells) or

a non-p53 mediated pathway. Investigation of the responsible endocytic pathways

may be pursued as a future direction. When our toxicity onset levels are consid-

ered in comparison with other literature using the same core-shell structure and

surface chemistry of QDs in keratinocyte cell lines, the data compares well. We

have tested the same QDs and surface chemistries used in these experiments with

the immortalized HaCaT keratinocyte line and found toxicity onset at a similar

concentrations (data not shown). Ryman-Rasmussen et al. have worked with

human epidermal keratinocytes (HEKs), an immortalized line, and found similar

results with the onset of toxicity for carboxylic acid coated 565 nm emitting and

655 nm emitting CdSe/ZnS QDs to be between 2.0 nM and 20.0 nM at 24 and

48 hours. [37] The cytotoxicity levels found in primary keratinocytes are in line

with the literature for ZnS capped and carboxylic acid functionalized cadmium

based QDs, as summarized by several sources. [6,7,9,54,55] Despite toxicity issues,

researchers have demonstrated QD tracking in live cells for over a week [56] and in

whole animals for several years. [57] However, in highly proliferative cells such as

basal keratinocytes non-apoptotic reactive species stress may be of greater con-


1

10

100

1000

0.5nM 5.0nM 50.0nM[QD]

Relative QD FluorescenceRel

ativ

e Q

D F

luor

esce

nce

Low Calcium No UVLow Calcium UVHigh Calcium No UVHigh Calcium UV

*

*

*

*

*

**

**

**

****

****

Figure 4.5: Primary Keratinocyte QD UptakeAn increasing amount of QD uptake is observable with increasing QD dose forboth proliferative and differentiated keratinocytes. QD uptake or association withproliferative and differentiated keratinocytes can be clearly seen using the averageof 4 separate experimental medians for each. The increase in noise at the high50 nM dose is likely due to saturation of cellular ability to uptake QD. The linesrepresent statistical significance between sample means at a single dose and theindividual marks represent statistical significance over control (∗ = p < 0.05,∗∗ = p < 0.01, ∗ ∗ ∗ = p < 0.001).


cern than cytotoxic events, as reactive oxygen species are well known to damage

DNA, and accumulation of these events could allow for genetic transformation. [58]

A forthcoming study by our lab not included as part of this thesis examines the

toxicity route in greater detail by investigating reactive oxygen generation re-

sponse to QDs, which is commonly thought to be a result of the catalytic action

of degrading nanoparticles inside the cell. [59]

Due to the possibility for environmental insults in combination with nanoma-

terial exposure, such as sun exposure along with nanoparticle containing cosmetic

application, the combination of external stress and QD or other NP exposure is

of interest. However, very few of such studies exist in the literature. One notable

example is a recent study by Chang et al. that examined the impact of UVA on

pancreatic carcinoma cells that were pre-exposed to thiol-capped CdTe QDs. [29]

They were able to find a substantial increase in cytotoxicity when cells were incu-

bated with QDs and then exposed to UVA. This data is interesting, but the cell

type, the pre-exposure with QD at high molarities, the use of UVA wavelengths,

and the time frame of QD exposure (4 hours maximum) limit comparison with our

study. Chang et al.did find that with shorter pre-exposure to QDs there was much

less impact on the QD toxicity, which suggests that their cytotoxic effect may be

due to internalized or degraded QDs non-radiatively releasing energy by catalyzing

reactive oxygen species generation. The research presented in this chaper isolates

the effects of UVB and QDs and examines their impact in primary human ker-

atinocytes, the cell type most likely to encounter UVB exposure in humans. We

find that UVB exposure does not change QD toxicity onset levels for either prolif-

erative or differentiated keratinocytes, despite the pre-sensitization and induction

of apoptotic pathways [60] and generation of dimeric pyrimidine photoproducts. [61]

The fact that we do not see a difference in the onset of toxicity for QDs suggests


that the mechanism of action of QD toxicity overwhelms the difference in UV

response.

When considering the uptake of QDs as determined by flow cytometry, the

difference between proliferative and differentiated keratinocytes is put into stark

contrast. At each QD dose, the difference in uptake between differentiated and

proliferative keratinocytes is statistically significant. The profound biological dif-

ferences between proliferative and differentiated keratinocytes, particularly the

increased presence of EGFRs [31–33] and low density lipoprotein receptors [34,35] in

proliferative keratinocytes may be in part responsible for the difference, as the

clathrin-coated pit related pathway of endocytosis of these receptors is known

to be a strong contributor to nanoparticle endocytosis in the size range of our

QDs. [45,62,63] These results are supported in part by Zhang et al. in their investi-

gation of carboxylic acid functionalized QD uptake in HEKs, who found low den-

sity lipoprotein receptors to be an important governing agent of QD uptake. [39]

It should be noted that they did not see any effect of clathrin inhibition, the

commonly associated internalization mechanism for low density lipoprotein re-

ceptors, [34,35] and that their earlier work suggested the ability of other agents such

as genistein- a known EGFR protein tyrosine kinase activity inhibitor [64]- to block

keratinocyte endocytosis effectively. [38] Clearly, more work is required to under-

stand the difference in uptake observed between proliferative and differentiated

keratinocytes, and several compensatory pathways may be involved. [45] Despite

the signaling pathway ambiguity, our findings are of great importance as the abil-

ity for proliferative keratinocytes to uptake high quantities of QDs while retaining

similar levels of viability raises concerns about potential cell death, dysfunction,

or transformation in highly proliferative non-apoptotic cells. The experiments

presented in this chapter of my dissertation provide additional evidence of the


ability of keratinocytes to endocytose QDs, and an interesting step forward in the

study of cellular targeting.

4.5 Conclusions

Consideration of the factors that govern QD interaction with primary keratinocytes

has yielded interesting results that suggest an ability of highly proliferative ker-

atinocytes to uptake much higher amounts of QDs than when in a differentiated

state. Additionally, we have found an intriguing lack of difference between the

uptake and cytotoxicity of QDs with and without pre-exposure to UVB. These

results suggest future directions to examine pathways that govern the observed

phenomena, as well as comparison with UVB and differentiation state effects of

titanium dioxide and zinc oxide, NPs that are more commonly used on skin. Im-

portant concerns have been raised in this chapter that suggest an increased risk

of mutagenesis and toxicity in the stem cell-like proliferative keratinocytes in re-

sponse to NPs. The consideration of NP interaction with the skin and localized

cell types is an important issue that warrants further research as human risk of

exposure increases.


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169

Chapter 5

Near-IR QD Confocal Imaging

The design, validation, andimplementation of a combined reflectanceand fluorescence mode near-IR confocalmicroscope for the imaging of QDs inmammalian skin 1

1Portions of this chapter are adapted from Mortensen, L.J., Glazowski, C.E., Zavislan, J.M.,and DeLouise, L.A. Near-IR fluorescence and reflectance confocal microscopy for imaging ofquantum dots in mammalian skin, submitted to Biomed Opt Express 2011.

CHAPTER 5. NEAR-IR QD CONFOCAL IMAGING 170

5.1 Introduction

As has been examined throughout this thesis, the disruption of skin barrier func-

tion, such as can occur with exposure to UVB, is able to increase the skin pene-

tration and local epidermal cellular interaction of quantum dots (QDs) and other

nanoparticles (NPs). [1–6] Since most NPs used commonly are above the size thresh-

old for renal filtration, [7] studies have suggested their ability to remain in the

body [8] and potentially to cause long term problems for a variety of NPs in many

cell types. [9–11]

The field of NP skin penetration is largely built on research into the percuta-

neous absorption of other substances. Evaluation of percutaneous absorption has

been a subject of investigation for a number of years, and the field of transder-

mal drug delivery has encouraged the development of efficient detection systems

for molecular targets, including analysis of acceptor solution in a static or flow-

through diffusion cell, in vivo systemic distribution, histological analysis, and

whole-tissue imaging in the skin. [12–14] Similarly, as interest in the diffusion of

NPs through the skin has grown, so too has the technology that endeavors to un-

derstand these phenomenon. However, when attempting to evaluate toxicological

effects of skin penetration, low frequency events may be important and so factors

such as detection limits and the state of the nanoparticle (i.e. dissolved ions or

intact NPs) in the skin become increasingly critical.

One of the most promising ways to address these problems is through devel-

opment of novel imaging modalities. For imaging of NPs, the possible modalities

depend on the physical characteristics of the NP. Fluorescent confocal microscopy

has been implemented by Richard Guy’s group to allow the whole tissue imag-

ing of fluorescent polymer NPs, and they have observed minimal levels of skin


penetration with intact skin barrier for hydrophilic and hydrophobic NPs. [15–18]

Work presented earlier in this thesis used whole tissue confocal microscopy as a

supporting technique to observe green fluorescent QD collection in the skin and

hair follicle openings without appreciable skin penetration in intact skin. Lopez et

al. have also used whole tissue confocal microscopy as a supporting technique, in

their case for the evaluation of QD skin penetration with sonophoresis and sodium

lauryl sulfate treatment. [19] One reason that whole tissue confocal microscopy has

served primarily as a supporting technique is the use of shorter visible range

wavelengths for excitation and emission, which limits observation depth. Mul-

tiphoton microscopy overcomes the excitation and average power limitations of

visible range confocal microscopy by using femtosecond pulses of near-IR light at

twice the excitation wavelength, thereby achieving greater imaging depth in the

skin. As mentioned in the introduction to this thesis, second harmonic generation

(SHG) of ZnO NPs using this technique has become common in the NP skin liter-

ature. [20–22] Kuo et al. have demonstrated the usability of SHG by evaluating the

impact of chemical penetration enhancers on the skin penetration of ZnO NPs. [23]

These studies are significant, as ZnO is an important particle that is commonly

used in topically applied sunscreens, but its commonly used dominant emission

peak at 385 nm limits the detection depth achievable. To attempt to address these

limitations, other techniques such as optical coherence tomography and coherent

anti-stokes raman spectroscopy have been implemented, but little literature exists

on their application in the skin. [24–26]

The work presented in this chapter examines the potential of a deep red exci-

tation source and near-IR emitting QDs to evaluate whole tissue skin penetration

of NPs. QDs are a category of NPs that have shown potential as a model for other

hard insoluble metallic and metal oxide NPs, and are of primary interest in the


electronics and biomedical research fields. They provide advantages in biomed-

ical imaging due to their broad excitation band, high photobleaching threshold,

ease of functionalization, and stability. However, no studies currently exist in the

literature that utilize whole tissue microscopy to localize the penetration profiles

of near-IR QDs through the skin. Additionally, despite work determining the

whole-body distribution of functionalized near-IR QD and targeting towards tu-

mor tissues, [27–29] we are unable to locate any studies that examine near-IR QD

skin penetration profiles and the impact of skin barrier status on skin penetration

using any techniques. The system reported in this section of this dissertation

endeavors to overcome the limitations of visible range whole tissue microscopies

by establishing a completely near-IR confocal microscopy system. We have de-

veloped an optimized near-IR excitation and emission confocal system that has

been fully characterized and validated to allow the imaging of QDs through the

epidermis and demonstrated its sensitivity through an ex vivo human skin sample.

Our system is shows potential to allow the evaluation of skin penetration profiles

for near-IR QDs and increase understanding of NP penetration mechanisms and

translocation through skin.


5.2.1 Instrumentation

For these experiments, a Lucid VivaScopeTM reflectance confocal microscope pro-

totype (Lucid, Inc., Rochester, NY) was rebuilt to allow imaging using both 660

nm and 785 nm laser wavelengths. Our schematic highlights the important compo-

nents of the system (Figure 5.1). The inclusion of a 664 nm long pass filter (LP02-


664RS, Semrock Corp., Rochester, NY) allows the detection of QD while excluding

the excitation wavelength. To maximize sensitivity, lenses with a visible to near-

IR coating (VIS-NIR coating, Edmund Optics, Barrington, NJ), fold mirrors with

a protected silver coating (ER.2, Newport Corp., Irvine, CA), an enhanced alu-

minum polygon (07 coating, Lincoln Laser Corp., Phoenix, AZ), a dichroic mirror

to combine beam paths (LaserMUX 659, Semrock Corp., Rochester, NY), and a

long wavelength silicon avalanche photo-diode (APD) detector (C5460/S8890 cus-

tom module, Hamamatsu Corp., Bridgewater, NJ) were selected as determined by

the manufacturer’s specifications. Scanning, timing, and collection systems used

were based on designs reported previously. [30] The 660 nm and 785 nm illuminat-

ing laser beams were scanned across the sample by the polygon and galvanometric

mirrors and relayed into the objective, a 30x 0.9-NA water immersion objective

(Photon Gear, Rochester, New York), with 5x magnification. The reflected and

fluorescently excited light was then relayed back out through the pinhole (138 µm

diameter) and to the APD. The effective field of view for our 10 bit 976x980 pixel

image is 0.319 mm x 0.323 mm, yielding a pixel pitch of 0.327 µm x 0.329 µm per

pixel. Axial scanning is performed using a micrometer to move the stage in the

z-direction. To determine system resolution, a technique developed in previous

studies to achieve Nyquist sampling and a smooth modulation transfer function on

a single edge target was used. [31] Using this technique, we found lateral and axial

resolutions of 0.6±0.02 µm and 4.8±0.15 µm for the 660 nm laser and 0.8±0.03

µm and 2.4±0.08 µm for the 785 nm laser.


660 nmlaser

785 nmlaser

Pinhole

Polygon

SiliconAPD

Objective

Dichroic mirror

Sample

Computer

Polarizing SplitterFluorescence

FilterGalvo

Figure 5.1: System SchematicThe basic optical design for the system used in these experiments. Reflectance(785 nm) and fluorescence (660 nm excitation) sources are pumped into a laserscanning confocal microscopy system.


5.2.2 Estimation of Sensitivity

The performance of a fluorescence imaging system in the skin depends on a num-

ber of factors. Here this thesis accounts for the known optical parameters in

order to reach a prediction of sensitivity. Benchmarking sensitivity measurements

by theoretical approximation of expected signal is an important task when ap-

proaching an imaging problem where an unknown concentration of a substance in

a biological milieu is the analyte. To achieve this for our system, the performance

of each system component is measured or estimated from manufacturer specifica-

tion and the reflectivity or transmitivity multiplied together at the 660 nm and

785 nm wavelengths. As such, the expression that includes the lens entry and

exit transmitivity (TLλi), the mirror reflectivities (RMλi), the dichroic mirror re-

flectivity (RDλi), the galvo reflectivity (RGλi), the polygon reflectivity (RPλi), the

polarizing beamsplitter surface transmitivity (TPB), the polarizing beamsplitter

internal surface reflectivity (RPB), the objective pupil area (AP ), the magnified

beam area at the pupil (AB), and the objective transmitivity (TOb) provides an

estimate of the transmitivity between the laser source and the focal plane (Equa-

tion 5.1). To condense the descriptive equation, each component factor is raised

to the power of its number of occurrences (lenses have an entry and an exit value)

in the beam path as depicted (Figure 5.1).

TSFPλi = (TLλi)8(RMλi)

4(RDλi)(RGλi)(RPλi)(TPB)2(RPB)(APAB

)(TOb) (5.1)

To test the accuracy of our estimates, we compared the predicted system

throughput with the actual laser power throughput for 660 nm in the system path

up to the focal plane. By testing at several powers, we found an average power


throughput at 660 nm of 18% before the objective, as compared with a prediction

of 19%. The minor disparity between our predicted and experimental values may

be due to some polarization rejection of the laser source. The theoretical objective

transmitivity is unreported by the manufacturer across our range of wavelengths,

but we found experimentally that the beam at 660 nm passed 17% of the laser

power present. These values are used in subsequent power approximations.

To estimate the ability of our system to measure light that returns to the

detector from a fluorescent probe, the noise equivalent power (NEP) of the de-

tector is used. NEP is a commonly quoted efficiency metric that describes the

needed amount of power to equal the inherent detector noise. For our detec-

tor, the manufacturer specified NEP is 0.15 pW/√Hz at the optimal sensitivity

wavelength of 940 nm normalized to a 1 Hz bandwidth modulation. The NEP is

proportional to the photo sensitivity, whose wavelength dependent characteristics

are reported by the manufacturer. Thus, a wavelength distributed NEP response

with relative values across the active range of our system (NEPDetectorλi) can be

estimated. This value must then be transformed for the bandwidth used in our

system, which requires division by the square root of the dwell time in seconds

(tdwell). The transmitivity of the system between the focal plane and the detector

(TFPDλi) is due to component efficiency, and increases the amount of power that

QD in the focal plane must generate to achieve NEP.

TFPDλi = (TOb)(TLλi)10(RMλi)

3(RGλi)(RPλi)(TPB)2(1

2RPB)

(NEPDetectorλi)√tdwell

(5.2)

The objective solid angle (Ω) is another important factor in the microscope

that decreases the portion of QD signal relayed through the system. QD emit light


isotropically in a classical dipole pattern, [32] and we assume random orientation of

the QD in the focal plane, which results in a uniform spherical average emission

(ΩSphere = 4π). However, the collection is limited by the numerical aperture of

the objective (NA) and the refractive index of the medium (RI), and scales the

collected QD fluorescence by the proportion of the objective solid angle to the

whole angle of the emission sphere.

Ω = 2π(

1− cos(

arcsin(NA

RI

)))(5.3)

Since the system and detector performance is strongly dependent on wave-

length, a realistic representation of the QD emission peak is used. For this pur-

pose, the manufacturer reported spectroscopic profile of our QDs results in a

semi-Gaussian distribution with a FWHM of 200 nm (Figure 5.2A). This normal-

ized profile is represented by the term IQDλi . For a specified peak wavelength, the

expression is numerically integrated (25 nm step size) from 200 nm to 1600 nm

using the corresponding values for components to yield the NEP needed at the

focal plane.

NEPQD =4π

Ω

λi=1600ˆ

λi=200

(IQDλi) (TFPDλi) dλi (5.4)

Additionally, since we wish to ultimately allow the imaging of QD through

epidermal tissue, the expression can be expanded to include the non-reduced tissue

scattering coefficients (µsλi), calculated from literature values for µ′sλi and g in

similarly sourced human skin, [33–38] and absorption coefficients (µaλi) from the

literature for ex vivo human tissue. [39] The non-reduced scattering coefficients were

selected based on wavefront error Strehl ratio comparison to scattering coefficients,


which found that the reduced scattering coefficients are too low to predict the

proper Strehl effect, and suggested that non-reduced scattering coefficients are

on the right order. A fixed depth (z) of 100 µm (Equation 5.5) was selected to

represent the average thickness of human epidermis and provides an estimate of

imaging ability through human epidermis.

NEPQD−epi =4π

Ω

λi=1600ˆ

λi=200

(IQDλi) (Tsystemλi)(e−z(µsλi+µaλi)

)dλi (5.5)

The next phase of the model is the determination of optimal laser wavelength

and the laser power needed to achieve NEP by exciting the QD with that wave-

length. We first selected a QD sample with a known concentration and a peak at

the optimal wavelength determined using our calculations, and measured the ab-

sorbance (Figure 5.2A). The Beer-Lambert law can then be used to determine the

extinction coefficient (ελ) at each wavelength, and this value substituted into the

equation established for use in CdSe/ZnS QDs [40] and validated in PbS QDs [41]

for a single QD absorption cross section (cm−2)

Cabs =2303ελNA

(5.6)

where NA is Avogadro’s number. To determine the laser power needed to

achieve NEP at the detector, the estimated QD power generation (Figure 5.3)

determined in Equation 5.5 is transformed into units ofW cm−2 using the absorp-

tion cross section (Cabs). For this portion of our estimate, the QD are assumed

to reside in the beam waist with a uniform illumination. With the inclusion of

the measured QD quantum yield (QY ) and proportion of fluorescence collected

in the dwell time, the laser fluence needed at the focal plane can be calculated.


To determine the proportion of QD fluorescence that is released during the dwell

time, the fluorescence lifetime is included using values from the literature for

the Evident Technologies PbS QDs used in our experiments. [42] Since the normal

excitation lag time is on the order of picoseconds, it is unnecessary to include

this in the calculation. Fluorescence emission is commonly modeled for QDs to

follow an exponential decay of the formula e−t/τ where τ is the fluorescence de-

cay, but for the Evident Technologies PbS QDs a two component fit of the form

A1 exp(−t/τ1) +A2 exp(−t/τ2) has been found to be more descriptive. [42] For our

samples, the established two component values from Hyun et al. will be imple-

mented, and so the proportion of signal collected becomes the integral of the decay

curve at the pixel dwell time divided by the integral of the decay curve as t→∞,

which we will refer to as IDT (0.24 in this case). The radius of the Airy Disk for

the laser is used to estimate the effective focused laser spot area, a function of the

objective numerical aperture (NA) and the laser wavelength (λL), to correct for

laser power density, yielding a laser power input distribution in units of W cm−2.

Since the absorption cross section of a QD is much smaller than the diffraction

limited beam waist focal spot, the area ratio is an important factor in determining

the relative amount of laser power in Watts needed at the focal plane (NEPLFP )

to achieve detector NEP.

NEPLFP =(NEPQDCabs

)(QY ) (IDT )

π (0.61λLNA

)2 (5.7)

To determine the power needed to achieve NEP at the laser source, theNEPLFP

(Equation 5.7) is then divided by the power loss from the focal plane to the source

as described above (Equation 5.1).


NEPLaser = (NEPLFP ) (TFPDλi)−1 (5.8)

To determine the impact of imaging through the skin, the NEPQD−epi term

expressed in Equation 5.5 is then substituted into Equation 5.7. This value is a

best-case scenario, since the Strehl ratio of the beam decreases with increasing

imaging depth in tissue.

NEPLFP−epi =(NEPQD−epi

Cabs

)(QY ) (IDT )

π (0.61λLNA

)2 (5.9)

The expression can be finalized in the same form as Equation 5.8 with the

additional inclusion of skin scattering (µsλi) and absorption (µaλi) through 100

µm of epidermis (z) to yield an increase in the needed laser power.

NEPLaser−epi = (NEPLFP−epi) (TFPDλi)−1(e−z(µsλi+µaλi )

)(5.10)

5.2.3 Quantum Dot Imaging

Near-IR QD with a lead-sulfide core and an emission peak of 900 nm in toluene

were purchased from Evident Technologies Inc. (Albany, New York) for our imag-

ing studies. The absorbance profile and quantum yields were measured to ensure

precise prediction of the system response to the QD (Figure 5.2A). To allow the

imaging of QD in a vertical configuration in solution, PDMS microwells (100

µm diameter by 10 µm deep) were filled with QD at a series of concentrations

and clamped between a glass coverslip and a microscope slide (Figure 5.2B and

5.2C). This procedure excluded the QD solution from the surrounding areas and

allowed background signal to be collected in the same frame as the QD signal and


separated using signal processing in Matlab (Figure 5.2D). Before each imaging

session, the laser power was calibrated to ensure accurate measurements. For

imaging through a separated epidermis, the same setup is used with the addition

of an ex vivo human epidermis between the PDMS microwells and the coverslip

that we separated as described below.

5.2.4 Skin preparation

Ex vivo human skin epidermis samples were obtained fresh from de-identified

healthy adult donors following abdominoplasty or mammoplasty (Strong and

Highland Hospitals, University of Rochester, NY), and stored at 4C. Usage was

approved by the University of Rochester Research Subjects Review Board. Within

6 hours of the surgical procedure, skin samples were rinsed with sterile 1x phos-

phate buffered saline (PBS), treated with 0.4 mL fungizone (Invitrogen) in 500

mL sterile 1x PBS for 10 min, and rinsed again thoroughly with 1x PBS. To allow

the diffusion of our epidermal separating agent, subcutaneous fat was removed

and the dermis thinned. The skin samples were then transferred to fresh 100 mm

sterile tissue culture plates with gauze and incubated overnight at room temper-

ature in 12 mL of 0.25% Dispase (Gibco Inc.) in a sterile cell culture hood with

the stratum corneum exposed to the air. The epidermis was then separated from

the dermis using tweezers and used for imaging immediately. To demonstrate

instrument proof of principle in skin, 30 µL of 10 µM PbS QDs in toluene were

applied to full thickness ex vivo human skin tape stripped (20x, Scotch 3M 3750

clear packing tape, USA) as described previously, [43] incubated for 24 hours with

skin viability maintained by sitting on a KGM-SF (Gibco Inc.) soaked gauze pad

with the stratum corneum exposed to the air, and used for imaging immediately


A.U

.

Wavelength

0

0.2

0.4

0.6

0.8

1

1.2

1.4

500 600 700 800 900 1000 1100 1200 1300

A B

C Diii

iii

Figure 5.2: Experimental SetupPictorial representation of an example normalized emission and absorbance curves(A) for the QDs used in these experiments. To enable imaging in the uprightposition, the PDMS microwells (B) of 100 µm diameter and 10 µm depth are filledwith QD and clamped as shown (C). Clamping the QD filled PDMS microwells(ii) between coverglass (iii) and a microscope slide (i) allows the imaging of afilled well. The QD signal (λmax=900 nm) can quite clearly be separated from thePDMS well fluorescence background (D).


thereafter.

5.3 Results and Discussion

5.3.1 Sensitivity Estimates

The predictions of system performance described above are useful in the estimation

of anticipated sensitivity and determination of realistic limits for a given technique.

With implementation for our detector and model QD emission distribution, the

minimum NEPs calculated using Equation 5.4 for QDs alone and Equation 5.5 for

QDs through 100 µm epidermis yield NEP minimums at 875 nm peak QD emission

(Figure 5.3A). This optimal value is close to that of the detector sensitivity, but

the steep fall-off of the detector (1100 nm cutoff) and the 200 nm FWHM of the

QDs push the detection values to slightly shorter wavelengths. Across the full

wavelength range of our system, necessary power to achieve detector NEP ranges

over 5 orders of magnitude (Figure 5.3A inset). When an absorbance curve for

QDs of this peak emission is converted to absorption cross section and included

with the losses from objective solid angle, focal spot size, and system components,

the resultant power needed to achieve NEP for a single QD can be estimated

using Equation 5.8 for QDs alone and Equation 5.10 for QDs through 100 µm

epidermis (Figure 5.3B). We plot the value from 200 nm to 1600 nm, and find a

clear minimum power needed to achieve detector NEP in the range of 600-700 nm

(Figure 5.3B). Thus, a laser line of 660 nm wavelength is selected. Our estimates

suggest that the necessary laser power at the source to observe a single QD is 5

Watts, with 7.3 Watts needed to detect a single QD below the thickness of a human

epidermis. Such a high laser power is problematic due to cost, safety concerns, and


1.E-06

1.E-05

1.E-04

1.E-03

1.E-02

600 650 700 750 800 850 900 950 1000 1050 1100

QD power

QD power throughepidermis

0

100

200

300

400

500

600

700

800

575 625 675 725 775 825 875 925

Wat

ts

Wavelength

Laser power Laser power through epidermis

1.E-061.E-051.E-041.E-031.E-021.E-011.E+001.E+011.E+02

200 400 600 800 1000 1200 1400 1600Wat

ts

Wavelength

1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 1E+10

200 400 600 800 1000 1200 1400 1600

A B

C

0.01

0.10

1.00

10.00

0 10 20 30 40 50 60 70 80 90 100

Lase

r Pow

er (W

)

QD Number

Laser Power to AchieveDetector NEPLaser Power to AchieveDetector NEP ThroughEpidermis

Figure 5.3: Model QD ResponsivityEstimation of the power needed to be generated by QD in the focal plane (A).The noise equivalent power (NEP) for the detector is scaled by the peak widthof the quantum dots, the collection angle, and the system components. A clearminimum is observable in the ∼900 nm wavelength emission peak range. Usingthe measured absorbance to estimate the absorption cross section then allows theminimum laser power needed to achieve NEP on a single quantum dot (note: inpractice this is limited by blinking) at a range of laser wavelengths, and it isobserved that there is a power minimum at the 600-700 nm wavelength range(B), for which a 660 nm laser line was chosen. When the power limitations of ourexcitation source is included, the system response to a range of QD concentrationsis able to be estimated (C).


destruction of the tissue sample. However, single fluorophore imaging is not vital

to the goals of whole tissue skin penetration imaging and is not commonly achieved

in other techniques. To provide an estimation of the minimum number of QD in

the focal plane needed to achieve NEP with varying laser power, Equation 5.10

is divided by the number of QD in the image plane for a given laser wavelength

and plotted (Figure 5.3C). The graph demonstrates that our laser currently in

use (135 mW at 660 nm) provides greater than NEP signal with 38 QDs in the

focal plane, and 57 QDs in the focal plane through 100 µm epidermis. If these

values are normalized to the experimentally determined lateral resolution (0.59

µm at 660 nm) the diffraction limited spot size, sensitivities of approximately 5.7

fmol/cm2 of QDs on the slide and 8.7 fmol/cm2 QDs through 100 µm epidermis

are achievable. The total applied dose in QD skin penetration studies reported

earlier in this thesis (Chapter 3) has been on the order of ∼ 17 pmol/cm2, which

suggests that our microscope will be able to detect as little as 0.03% of the applied

dose on glass and 0.05% of the applied dose through 100 µm epidermis. If the

laser power is increased to 1.5 W, a value that is achievable using a krypton ion

laser, the number of QDs in the beam waist needed to reach NEP drops to 4 on

glass and 6 through 100 µm epidermis, which corresponds to 0.61 fmol/cm2 on

glass or 0.92 fmol/cm2 through 100 µm epidermis. These values correspond to

0.004% and 0.005% of the applied dose, respectively. However, this power input

would yield an average power at the focal plane of approximately 47 mW, which is

above the maximum permissible exposure (MPE) value of 20.0 mW for skin with

constant illumination over a spot with diameter less than 3.5 mm (ANSI Z136.1).

Using MPE to determine the maximum safe detection yields 10 QDs on glass and

15 QDs through 100 µm epidermis, or 1.47 fmol/cm2 on glass or 2.24 fmol/cm2

through 100 µm epidermis. This corresponds to 0.009% and 0.013% of the applied


dose, respectively. Direct comparison to existing techniques is challenging, as

few studies define their system sensitivities in the same fashion. However, our

predicted sensitivity establishes the viability of whole tissue confocal microscopy

in the near-IR as a technique with the potential to provide valuable information

about permeation of substances through the epidermis.

5.3.2 Experimental Validation and Model Comparison

To determine whether the system in practice holds up to its theoretical predictions,

measurements of fluorescence intensity from QDs at various concentrations have

been executed. As described in the materials and methods section, we suspended

various concentrations of QDs in PDMS microwells clamped tightly between the

coverslip and a microscope slide. The presence of 100 µm diameter microwells

enables the background to be calculated from the same image as the QD signal,

allowing for a facile representation of signal to noise within a sample. Each ex-

periment was averaged over 6 different microwells at each concentration, and the

curve repeated at 3 laser power levels (1.6 mW, 2.4 mW, and 3.9 mW at the focal

plane, data not shown). In the high laser power case (3.9 mW at the focal plane),

the signal gained from the QDs is distinguishable from background down to a con-

centration as low as 0.1 µM , and increases linearly over two orders of magnitude

to a concentration of 10.0 µM (Figure 5.4). When the QD are imaged through

separated human epidermis, there is a substantial decrease in the signal intensity

across a range of concentrations, with the lowest detectable concentration of QD

at 2.0 µM . The imaging of a known concentration of a fluorescent probe to de-

termine its limit of detection is an important task when attempting to determine

the ability of the a system to detect an unknown concentration of the probe in


0 2 4 6 8 10 120

20

40

60

80

100

120

140

160

180

200

QD in microwellQD through skinNoise in microwellNoise through skin

A.U

.

[QD] (µM)

Figure 5.4: Experimental QD ResponsivitySignal response of system to QD across a range of concentrations in the microwelland through ∼100 µm separated human epidermis with a laser power of 3.9 mWat the focal plane. Background noise is also plotted, and is calculated from thesame frames as the signal.


a tissue sample. Our technique of placing a known concentration of QDs behind

approximately 100 µm of human epidermis allows for an accurate determination

of the lower limit of detection in our system in practice, with the exception of

scattering that would occur from apical QDs residing in the upper layers of the

epidermis.

In order to compare our results to varied concentrations of QDs to our pre-

dictions, it is necessary to determine the predicted response to the number of

QD in a volume rather than the previously described area calculation. To do so,

the number of QDs detected are scaled to the focal volume as determined by the

lateral resolution (0.59 µm) and axial resolution (4.75 µm) of our system in prac-

tice. When plotted over the range of concentrations tested, the predictive measure

matches well to experimental data (Figure 5.5). For QDs imaged in the microw-

ell alone and through human epidermis, our experimental fluorescent intensities

matched expected values at low concentrations, but did not attain predicted inten-

sity at higher concentrations. This may be caused by the assumption of uniform

excitation efficiency of QDs in the focal plane, and the higher deviation from the

model present when imaging through human epidermis may suggest a limit of epi-

dermal scattering estimation or greater thickness of ex vivo separated epidermal

tissue than expected. Differences are relatively minor at all tested QD concentra-

tions. These values suggest that our system is operating at a near-ideal efficiency,

and inform a prediction of the ultimate limit of our sensitivity in practice.

Demonstration and calibration of the imaging system using QDs in microw-

ells through separated epidermis nominally verified the predicted system perfor-

mance. The next step is execution of skin penetration evaluation using topically

applied QDs. To this end, QDs at stock concentration were applied to the skin


1

2

3

4

5

6

7

8

9

10

Rel

ativ

e Fl

uore

scen

t int

ensi

ty

0.1 1.0 10.0

[QD] (μM)

QD actual valuesQD actual values through skinQD theoretical valuesQD theoretical values through skin

Figure 5.5: Model and Experimental System ResponsivityThe behavior of the experimental data follows the model. The improved theoret-ical sensitivity is expected, as we use idealized versions of the laser beams, QDs,and other components as well as scattering and absorption coefficients from theliterature.


in their toluene vehicle. Toluene is known to penetrate mammalian skin in high

levels, [44,45] and a 24 hour application procedure of QDs in organic solvent with ad-

ditional barrier disruption through mechanical tape-stripping disruption provides

an experimental model for potential incidental exposure to QDs in the workplace

through damaged skin. Results find that skin images from the reflectance channel

are strongly degraded even close to the surface of the skin (Figure 5.6A), but cellu-

lar borders can still be observed (red arrows). QD fluorescence evaluation yields a

strong signal (Figure 5.6B) that can be traced into the tissue depth (Figure 5.6C).

A much slower drop-off in signal is clear versus the reflectance in depth, and when

the ratio of these numbers is determined, the effective flux rate is plotted. Addi-

tional noise is present when imaging deeper in the tissue, but the curve suggests

partitioning of the toluene-containing QDs at the stratum corneum/ epidermal

transition and a steady diffusive release into the epidermis. This steady diffusion

profile implies that strong enough barrier damage can induce diffusive behavior

of NPs, as opposed to the seed point model discussed earlier in this thesis with

UVB barrier disruption. When the QD fluorescence and skin reflectance profiles

are averaged over 6 locations in the skin (Figure 5.6D), a consistent presence of

QD into the epidermis is clear, confirming the ability of our system to evaluate

the skin penetration of QDs under relevant experimental conditions.

Since fluorescence is one of the key mechanisms that has been used to evaluate

the skin penetration of NPs, understanding the technical limitations of evaluation

techniques has important real-world implications in the application of results to

assess risk and potential systemic toxicology. Noticeably lacking from all but the

most rigorous of publications is an estimation of the minimum number of NPs

that must be present to be detectable, and none that we have been able to find


0

0.2

0.4

0.6

0.8

1.0

1.2

0 5 10 15 20 25 30 35 40 45

Rel

ativ

e Fl

uore

scen

t Int

ensi

ty

Tissue Depth (µm)

QD Fluorescence SignalSkin Reflectance Signal

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25 30 35 40

Arb

itrar

y U

nits

Tissue Depth (µm)

QD Fluorescence SignalSkin Reflectance SignalSignal Response Ratio

A B

C D

Figure 5.6: QD Skin PenetrationSkin penetration of 900 nm peak emission PbS QDs in toluene. The reflectancesignal close to the surface of the skin is degraded by the highly scattering QDs andtoluene barrier disruption (A). However, some cell borders can still be resolved(arrows). The QDs provide a strong fluorescence signal at the same plane (B).When these profiles are plotted, a clear permeation of QDs into the epidermis isobservable relative to the collected reflectance signal (C). The ratio of these valuesat each dose provides a steady-state flux curve determination, whose shape sug-gests that there is some partitioning of the toluene solvated QDs at the stratumcorneum/epidermal junction with a steady release through the epidermis there-after (C). The average of 6 measurement locations yields a curve exhibiting theaverage permeation of PbS QDs into the epidermis (D).


utilize fluorescence microscopy except as a supporting technique. In fact, only one

study that we are currently aware of has been able to use whole tissue microscopy

to determine a skin penetration profile of NPs past the stratum corneum. Kuo et

al. have used second harmonic generation with multiphoton microscopy to find a

change in the penetration profiles of ZnO NPs with various chemical penetration

enhancing agents. [23] Their studies suggest that the treatment of skin with acetone,

oleic acid, and a mixture of both is able to increase the collection of ZnO NPs

in the stratum corneum and their diffusion through the epidermis. Use of SHG

enables the distinction of NPs from released ions, an advantage that it shares

with fluorescence. Despite this important advancement in the usage of whole

tissue microscopy as a primary skin penetration evaluation technique, the authors

did not address their ultimate sensitivity. To find published articles that examine

the ultimate sensitivity of their techniques, more overtly quantitative techniques

based around mass spectroscopy must be considered. Research by Gopee et al.

has investigated the penetration of QD though intact and damaged murine skin

in vivo, and used the analysis of Cd in distal organs (liver and lymph nodes) to

evaluate skin penetration. [5] Their study provides the limit of detection and the

limit of quantification for their technique, but suffers due to the necessity of a large

portion of QDs to penetrate the skin in order to achieve appreciable collection in

the distal organs over background, with the first detectable difference in barrier

defect occurring after 2% of the applied dose collected in the liver. In a previous

study they injected QDs subcutally- mimicking the penetration of 100% of an

applied dose- and found only 6% of the applied dose in the liver and 1% in the

regional draining lymph nodes. [46] Hence, the liver collection levels required for

their skin penetration study suggests that skin barrier disruption must allow 33%

of the applied dose to penetrate to a subcutal level for detection. This value is


well within the ability of our system to evaluate. Recent work by Lopez et al. has

studied the ability of sodium lauryl sulfate (SLS) and ultrasound to synergistically

impact barrier function against QDs. [19] A very small but quantifiable percentage

of the applied QDs were found to penetrate intact epidermis (0.006%-0.078% of

the applied dose) by mass spectroscopy on mechanically separated and cleaned

dermis. The suggested pathway for their observed QD penetration is via lacunar

imperfections of approximately 48 nm diameter and covering 0.44% of the skin

surface area, as discussed in previous work by Mitragotri’s group. [1] If it is assumed

that the majority of QD skin penetration occurs through these pathways, then

it can be estimated that the localized penetration channels allow 1.36%-17.73%

of the applied dose to pass, which is within the detection limit of our system,

and their ultrasound/SLS treatment increases the penetration of QDs into the

dermis to levels of 80%- 99% of the applied dose. Lopez et al. calculated the

limit of detection and quantification in a similar manner to that of Gopee et al.,

but baseline level of Cd observed in the dermal samples with no QD application

was not reported. Whole tissue visible range confocal microscopy of the separated

dermal samples supported their mass spectroscopy findings, with rare occurrences

of localized spots of relatively high QD concentration, but was not used to establish

a diffusion gradient. Results reported earlier in this thesis (Chapter 3) found that

UVB exposure increased QD penetration with liver collection of Cd that equaled

0.0073% of the applied dose. If the 6% liver distribution of dermally penetrated

QDs figure is used from Gopee et al., [46] the amount of dermally penetrated QDs

would be well within the detection capabilities of the confocal system presented

in this chapter. One caveat when estimating the fluorescence signal from mass

spectroscopy is that the technique is unable to distinguish between the penetration

of intact NPs or dissolved ions. With an acute application dose of ZnS-capped QDs


this may not be a major concern, but will be an important factor with other types

of NPs, such as silver or ZnO NPs. [47,48] The sensitivity evaluation of our system

and practical application for imaging through human epidermis provides a distinct

advantage for future studies to determine the impact of skin barrier disruption

on NP skin penetration and establish a model to understand the penetration of

nanoparticulate substances.

Conclusions

The assessment of NP skin penetration, either to determine risk factors or provide

targeted delivery is an important area that has far-reaching clinical and toxicolog-

ical implications. This chapter’s development of a whole tissue confocal imaging

system in the near-IR range is a promising technique that exhibits great poten-

tial to address some previous limitations in the field, and to complement existing

state of the art whole tissue imaging techniques and quantitative techniques. We

have optimized and characterized our reflectance and fluorescence confocal system

performance and found the ability to image QDs in a controlled fashion through

ex vivo human epidermis, with sensitivity surpassing 0.3% of our applied dose,

and implemented it to detect QDs penetrating the skin in an ex vivo barrier dis-

rupted model. Application optimized system design and thorough testing enables

greater confidence in imaging results and has the potential to expedite the eval-

uation of NP formulations and skin barrier alterations. With the movement of

imaging modalities into the near-IR, a well characterized system can provide in-

creased sensitivity and detection depth for an improved understanding of NP skin

permeability.


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202

Chapter 6

Conclusions and Future Directions

CHAPTER 6. CONCLUSIONS AND FUTURE DIRECTIONS 203

With the revolutionary promise proffered by the unique characteristics of

nanoparticles (NPs) growing closer to actualization, understanding their impact

on the human system is ever more critical. The work presented in this thesis

has addressed the interaction of quantum dot nanoparticles (QDs) with the skin,

one of the highest risk organs for environmental interaction with QDs and other

NPs. NPs are commonly used in sunscreens and other cosmetics, and since con-

sumers may apply sunscreen to sun damaged skin, the effect of UVB on NP skin

penetration raises potential toxicity concerns. QDs were selected as a model NP

in this thesis due to their advantageous fluorescent properties; and widespread

and expanding use in research applications and manufacturing provide additional

primary interest. This thesis has delved into the impact of UVB on skin barrier

function and QD skin penetration in vivo, has investigated the impact of UVB

and keratinocyte differentiation on QD uptake and toxicity, and has designed and

implemented an optimized reflectance and fluorescence confocal microscope to

advance technical evaluation in the field. This thesis has contributed novel and

relevant results to the scientific community, and provided important insight into

the impact of skin barrier status on QD skin penetration.

In the first experimental portion of this thesis, Chapter 2, the impact of UVB

on skin penetration of QDs applied for 24 hours immediately following irradiation

was evaluated. Carboxylated QD were applied to the skin of SKH-1 mice in a

glycerol vehicle with and without 270 mJ/cm2 UVB exposure. The skin collec-

tion and penetration patterns were evaluated 8 and 24 hours after QD application

using tissue histology, confocal microscopy, and transmission electron microscopy

(TEM) and EDAX analysis. Low levels of penetration were seen in both the non-

UVB exposed mice and the UVB exposed mice, with qualitatively higher levels

of penetration observable in the UVB exposed mice. These studies first demon-


strated the importance of UVB to affect the penetration of QDs (∼30 nm diam.)

in the in vivo SKH-1 mouse model. This was an important advancement as it

suggested that UVB induces an outside-in barrier defect likely due to a loss of

epidermal calcium gradient and resultant stratum corneum lipid disruption. Im-

portant trends in the collection of QDs in the folds, defects, and hair follicles

were also noted that may have combined with the weakened stratum corneum to

allow increased skin penetration. This chapter contains the first published exam-

ple of the impact of UVB on skin penetration of NPs, contributing an important

advancement to the literature.

To expand the observations in the previous section on the impact of UVB on

QD skin penetration, this thesis next pursued a designed experiment approach

to evaluate the impact of UVB on inside-out skin barrier function and optimize

study of UVB induced outside-in barrier function to QDs. Skin barrier disruption

as measured by transepidermal water loss (TEWL) was found to increase with

increasing dose of UVB in a delayed fashion that peaked 4-6 days after UVB,

depending on the dose. Carboxylated QDs were applied over the peak TEWL

time points, and their skin penetration evaluated using fluorescence microscopy,

TEM, and quantitative atomic absorption spectroscopy of the distal organs. A

qualitative increase in skin penetration after UVB exposure could be found using

microscopy techniques, along with a low level quantitative increase in the Cd levels

in the liver. Interestingly, statistically significant but still low levels of QD collec-

tion in the lymph nodes without UVB exposure was measured, whose magnitude

decreased with UVB exposure. These results constitute the first reported instance

of topically applied QDs trafficking to the lymph nodes in healthy animals. This

trafficking is decreased by exposure to UVB, presumably from the well-accepted

impact of UVB on Langerhans cell presence. Future work will focus on quantify-


ing and understanding associated risk factors with the suggested Langerhans cell

uptake observed after topical QD application, and could lead to novel therapeutic

or vaccine modalities. Additionally, further research into modeling and experi-

mentally verifying the “seed point” NP skin penetration model suggested by this

chapter could yield important understanding into risk factors for skin penetration

and mechanistic insight into the process. This chapter suggests that QDs can

be trafficked to the lymph nodes in healthy skin and UVB irradiated skin at low

levels, and that UVB quantitatively increases the liver collection of Cd after QD

application, thereby offering the first quantitative analysis of UVB impact on NP

skin permeation.

As determined in the previous sections of this dissertation, exposure of skin

to UVB can increase its QD permeability, and so the risk of their interactions

with the local epidermal cell types grows. In the next portion of this thesis, the

impact of QDs on proliferative and differentiated keratinocytes was considered.

Keratinocytes are the dominant skin cell type present in epidermis, and the varied

differentiation states of keratinocytes throughout the layers of the epidermis from

the stratum basale up through the stratum spinosum and granulosum strongly af-

fect cellular stress response and endocytic abilities. In this chapter, the differences

in acute QD cytotoxicity and uptake of carboxylated CdSe/ZnS core/shell QDs

between proliferative and differentiated primary keratinocytes with and without

UVB exposure was evaluated. These in vitro studies serve as a model system to

approximate the effects of UVB and NP interaction with keratinocytes throughout

the human in vivo epidermis after UVB exposure. A dependence on keratinocyte

differentiation state for UVB toxicity was observable, but UVB exposure did not

impact the toxicity or cellular uptake of QDs. Additionally, keratinocyte differen-

tiation state strongly impacted the cellular uptake of QDs but had no discernible


effect on toxicity values. These findings suggest that keratinocyte differentiation

state is an important factor in the risk of keratinocyte QD uptake, and that this

cellular uptake may increase risk of downstream cellular transformation in the QD-

positive live fraction of stem cell-like long-lasting basal proliferative keratinocytes.

Important questions are raised about the endocytic pathways responsible for QD

uptake in proliferative and differentiated primary keratinocytes, and the possibil-

ity of targeted NP delivery to these cells.

The final portion of this thesis has used an optimized near-IR whole tissue

microscopy system to take steps towards overcoming the sampling error and sen-

sitivity challenges in the evaluation of NP skin penetration that have limited appli-

cability and reproducibility of results in the literature. The system presented has

built on the promise offered by current literature using confocal and multi-photon

microscopies to evaluate whole tissue skin penetration of NPs. In this chapter,

an optimized whole tissue confocal microscopy system was designed, built, and

validated to allow imaging of near-IR lead sulfide QDs through ex vivo human

epidermis. A 785 nm reflectance source and 660 nm fluorescence excitation source

are used to provide morphological information and QD fluorescence evaluation

respectively. The system has been demonstrated to allow imaging of lead sulfide

QDs through a separated human epidermal scatterer, the system response curve

evaluated over a range of concentrations, and the skin penetration of PbS QDs

through a damaged barrier measured. System sensitivity rivals other state of the

art techniques, and a path forward to increase sensitivity by increasing input laser

power has been suggested. This chapter has validated that near-IR fluorescence

confocal microscopy can be used to evaluate skin penetration of QDs. Future

work will further implement the technique to provide more precise and expedited

evaluation of QD formulations and skin barrier disruption.


In summary, this thesis has yielded important and novel results that have con-

tributed to the growing body of NP skin penetration literature. Results are the

first published to investigate the impact of UVB on NP skin penetration and some

of the first to quantitatively evaluate the skin penetration of QDs. This disserta-

tion has evaluated the impact of UVB on QD skin penetration, the effects of UVB

and cellular state on QD cellular interaction, and provided technological advance-

ment through optimized confocal imaging. These results suggest important future

directions to more fully understand the impact of NP properties such as surface

functionalization and charge, NP size, and core composition (i.e. gold NPs vs.

QDs) on skin permeability. Additional factors such as repeated skin exposure to

NPs and UVB or work to determine the particulate or ionic state of NPs that pen-

etrate the skin also warrant investigation, and may prove to be important factors

in the determination of human health risk. Technical tools and collaborations to

enable these directions have been validated, including quantitative evaluation of

QD skin penetration and a whole-tissue near-IR confocal microscope. Findings of

this thesis can be directly applied to the formulation of NP containing cosmetics

to minimize skin penetration and guidelines for NP cosmetic topical application

after UVB exposure, and can be used to design NPs to increase trafficking to

lymphatic system or gross penetration levels. Additionally, future in vivo studies

using custom imaging modalities and commercial formulations could generate the

necessary insight to assess human health risks from application of NP containing

cosmetics to UVB-exposed skin.

208

Appendix A

Chapter 2 Supplementary Data

APPENDIX A. CHAPTER 2 SUPPLEMENTARY DATA 209

Particle Size Distribution COOH-QD in ddH2O Initial

0

5

10

15

20

25

30

0.46

0.83 1.

5

2.7

4.85

8.72

15.7

28.2

50.7

91.3

164

295

531

955

Particle Diameter (nm)

Mea

n N

umbe

r

20.1 ± 2.1nm

a.

Particle Size Distribution 75% Glycerol, 2 hr

0

5

10

15

20

25

30

0.4

0.72

1.29

2.33

4.19

7.53

13.5

24.4

43.8

78.8

142

255

459

825

Particle Size (nm)

Mea

n N

umbe

r

33.2 ± 2.8nm

b.

Particle Size Distribution

COOH-QD 75% Glycerol, 24 hr

0

5

10

15

20

25

30

0.4

0.72

1.29

2.33

4.19

7.53

13.5

24.4

43.8

78.8

142

255

459

825

Particle Size (nm)

Mea

n N

umbe

r

30.0 ± 3.1nm

c.

Figure A.1: Malvern Raw Particle SizeMalvern particle size raw data scans of QDs in (a) water, (b) 75% glycerol at 2hr and (c) 75% glycerol at 24 hr stored at room temperature.


Figure A.2: Epidermal Thickness Post-UVBEpidermal thickness data post UVB exposure (8 and 24 hr) relative to control (noUVB).


SC

Epidermis

40X

Figure A.3: Thickened Skin 4.5 Days Post-UVBBright field image of thickened skin at 4.5 days post-UVB


Figure A.4: QD Collection in DefectsCyrohistology section for 8hr ctrl (no UVR) in bright field (left) with correspond-ing fluorescence image (right) showing QD collection in stratum corneum defects(arrows) . Dapi – blue cell nuclei stain. Red is Loricrin a SC protein.


Figure A.5: Silver Enhanced Particle SizeSize of QDs that have been silver enhanced in the tissue.


Figure A.6: Negative Control TEM and EDAXTEM image of negative control (no QD, glycerol only) and corresponding EDAXspectral scan of dark deposits showing no evidence of silver deposition.

215

Appendix B

Chapter 3 Supplementary Data

APPENDIX B. CHAPTER 3 SUPPLEMENTARY DATA 216

male

female

0 mJ

180 mJ

270 mJ

360 mJ

t0t1t2t3t4t5t6t7

Figure B.1: ANOM Chart for TEWL


0 m

J/cm

2 U

VB27

0 m

J/cm

2 U

VB36

0 m

J/cm

2 U

VB

Figure B.2: UVB Peak Dose Morphology


0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

2.1

2.3

2.5

0 1 2 3 4 5 6 7 8 9Time (Days after Irradiation)

Nor

mal

ized

TEW

L

hump tail neck

Tail Neck

Hump

Figure B.3: Back Measurement LocationBack position TEWL response to 180 mJ/cm2 UVB over time. The TEWL mea-surements off of the peak back position do not show any change in TEWL.


Figure B.4: TEM Negative Control

110415 corrected thesis

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