Studies with multiple vitamins encapsulated in nanosomes for protection of skin from UV damage

179 Pages·2012·2.95 MB·English

by  Sunkari, Chetana

Studies with Multiple Vitamins Encapsulated in Nanosomes for Protection of Skin
from UV Damage

A Thesis
Submitted to the Faculty
of
Drexel University
by
Chetana Sunkari
in partial fulfillment of the
requirements for the degree
of
Doctor of Philosophy
December 2011
© Copyright 2011
Chetana Sunkari. All Rights Reserved.
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DEDICATIONS

This thesis is dedicated to my parents Dr. Ramakrishna Rao Sunkari and Mrs. Sujatha
Sunkari, and my late advisor Dr. Elisabeth Papazoglou. I am grateful to all of you for
being a part of my life and inspiring me to put my best into everything I do.
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ACKNOWLEDGEMENTS

I would like to thank my advisors Dr.Elisabeth Papazoglou and Dr. Andres Kriete for
their guidance and invaluable support. Dr. Papazoglou was a great role model, who
taught me many a life lesson, both personal and professional. Her enthusiasm and drive
was unparallel and if not for her this project would not have happened. Special thanks to
Dr. Kriete for taking me under his wing and supporting my work and this project after Dr.
Papazoglou’s passing.
I would like to thank my committee Dr. Fred Allen, Dr. Margaret Wheatley, Dr. Jouni
Uitto and Dr. Wan Shih for their extremely insightful thoughts and ideas on my project.
All of the comments were very important to bring a direction to this thesis.
I would like to thank my colleagues and friends that taught and helped me with my
experiments. I would like to thank Dr. Mike Neidrauer and Dr. Leonid Zubkov for all the
help with the skin optics, Dr. Anat Katsir for teaching me the basics of histology, Dolores
Conover for solving every lab related problem I ever had and Dr. Zhenyu Huang for all
the lunches. A big thank you to my friends Ranjita Bose, Rosemary Bastian, Brandon
Johnson, Ramalingam Venkat Kalyanasumdaram and John Alamia for being there
everytime I needed a break. I am also grateful for my colleagues and friends Joshua
Samuels, Josa Hanzlik, Raha Dastgeyb, An Nguyen, Xiang Mao, Dr. Greg Botta and
Diane Keene. My experience would here would not have been so amazing without you
guys.
I am extremely grateful to my friends Swapna Chittajallu, Vijay Harsha, Hari Kamaraju,
Pallavi Mallajosyula and Krishna Pinnamaraju for all the weekends we spent together. A
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special thanks to Madhuri Penmatsa for being such a good listener and friend and to Dr.
Vamshi Gangupomu for being such a great support and inspiration.
I would also like to thank everyone in the Department of Biomedical Engineering,
Sciences and Health Systems, Drexel University. Thank you, Natalia Broz for being so
amazing and such a great friend. Also, thank you to Lisa Williams, Danielle Crocker,
Frank Kepics and Steve Detofsky for helping out everytime I needed something. Thanks
to Dr. Banu Onaral for being extremely supportive.
Last, but definitely not the least, I would like to thank my family for their love and faith
in me. My mom Sujatha Sunkari, My dad Dr. Ramakrishna Rao Sunkari and my sister
Shwetana Sunkari - I am grateful to be a part of your family. You gave me opportunities,
unconditional love and immense support that I am very grateful for.
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TABLE OF CONTENTS
DEDICATIONS ................................................................................................................. i
ACKNOWLEDGEMENTS ............................................................................................. ii
TABLE OF CONTENTS ................................................................................................ iv
LIST OF FIGURES ......................................................................................................... xi
LIST OF TABLES ......................................................................................................... xvi
ABSTRACT ..................................................................................................................... xx
CHAPTER 1 : INTRODUCTION AND SPECIFIC AIMS .......................................... 1
1.1. Introduction .................................................................................................... 1
1.2. Project Description ......................................................................................... 4
1.3. Context and Rationale .................................................................................... 4
1.4. Specific Aims and Hypotheses ....................................................................... 5
CHAPTER 2 : BACKGROUND ..................................................................................... 7
2.1. Significance of solar UV radiation ................................................................ 7
2.2. Effects of UV exposure ................................................................................... 9
2.2.1. Sunburn and inflammation .................................................................. 10
2.2.2. Skin Cancer ............................................................................................ 10
2.2.3. Photoaging.............................................................................................. 11
2.3. Methods for UV damage detection .............................................................. 12
2.3.1. Introduction ........................................................................................... 12
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2.3.2. Diffuse Reflectance Spectroscopy ........................................................ 13
2.3.3. Skin Auto fluorescence.......................................................................... 15
2.3.4. Other non-invasive detection methods ................................................ 16
2.3.5. Histological evaluation of Solar UV Damage ...................................... 17
2.4. Solar UV radiation and treatments ............................................................. 18
2.4.1. Vitamin A ............................................................................................... 18
2.4.2. Vitamins C and E .................................................................................. 19
2.4.3. Other vitamins, vitamin-based treatments and other antioxidants .. 20
2.5. Molecular Markers of UV damage ............................................................. 22
2.5.1. Matrix metalloproteinase and Procollagen ......................................... 22
2.5.2. Antioxidants and Antioxidant Enzymes .............................................. 23
2.5.3. Reactive Oxygen Species ....................................................................... 24
2.5.4. Nuclear and mitochondrial DNA Damage .......................................... 24
2.6. Mechanisms of UV damage ......................................................................... 25
2.6.1. MAP Kinase Pathway ........................................................................... 26
2.6.2. Syk Kinase Pathway .............................................................................. 27
2.6.3. Apoptosis ................................................................................................ 28
CHAPTER 3 : AIM 1 – TO FIND THE OPTIMUM CONDITIONS FOR THE
MANUFACTURE OF NANOSOMES REQUIRED TO DELIVER DRUG TO
SKIN. ................................................................................................................................ 30
3.1. Objective ........................................................................................................ 30
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3.2. Materials ........................................................................................................ 32
3.2.1. Lipids ...................................................................................................... 32
3.2.2. Vitamins ................................................................................................. 32
3.2.3. Other Chemicals .................................................................................... 33
3.2.4. Other Materials ..................................................................................... 33
3.3. Methods ......................................................................................................... 33
3.3.1. Manufacture of Liposomes ................................................................... 33
3.3.1.2. Hydration Method ............................................................................. 33
3.3.1.3. Reverse Phase Evaporation Method (REV method) ...................... 34
3.3.2. Liposome Sizing and Dialysis ............................................................... 34
3.3.2.1. Extrusion ............................................................................................ 34
3.3.2.2. Probe Sonication ................................................................................ 35
3.3.3. Dialysis and Characterization .............................................................. 35
3.3.4. Diffusion Experiments .......................................................................... 36
3.3.5. Cryosectioning for CF extraction ........................................................ 36
3.3.6. CF Extraction ........................................................................................ 37
3.4. Results ............................................................................................................ 37
3.4.1. DOPC penetrates skin better than DPPC ........................................... 37
3.4.2. Optimum encapsulation/size index of drug is achieved by a
combination of rehydration-dehydration and sonication methods ................ 39
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3.4.3. Liposomes of 100nm size display the highest diffusion through skin
…………………………………………………………………………..41
3.4.4. Liposome penetration into the skin occurs within 2 hours ............... 43
CHAPTER 4 : AIM 2 – TO CORRELATE THE NON-INVASIVE OPTICAL
DATA WITH KNOWN BIOLOGICAL ASSAYS ON THE SKH1 HAIRLESS
MOUSE ANIMAL MODEL .......................................................................................... 45
4.1. Objective ........................................................................................................ 45
4.2. Materials and Methods ................................................................................ 46
4.2.1. Solar Simulator ...................................................................................... 46
4.2.2. Animal Model Experiments.................................................................. 47
4.2.2.1. Animal Model .................................................................................... 47
4.2.2.2. Animal Model and UV Radiation .................................................... 47
4.2.3. Diffuse Reflectance Spectroscopy (DRS)............................................. 50
4.2.4. Image analysis ........................................................................................ 51
4.2.5. Fluorescence Measurements................................................................. 52
4.2.6. Histology ................................................................................................. 53
4.2.7. Skin Protein Extracts and Western Blot Analysis ............................. 53
4.3. Results ............................................................................................................ 54
4.3.1. Erythema via image analysis ................................................................ 54
4.3.2. Blood volume levels increase over the period of irradiation as
compared to the controls .................................................................................... 56
4.3.3. Fluorescence changes induced by UV radiation ................................. 57
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4.3.4. Histological data show increases in epidermal thickness and
epidermal apoptotic keratinocytes .................................................................... 58
4.3.5. MMP-13 levels change with increased UV exposure ......................... 60
CHAPTER 5 : AIM 3 – TO DETERMINE THE EFFICACY OF INDIVIDUAL
VITAMINS AND THEIR COMBINATIONS IN VITRO .......................................... 62
5.1. Objectives and Introduction ........................................................................ 62
5.2. Materials and Methods ................................................................................ 62
5.2.1. Cell lines ................................................................................................. 62
5.2.2. Materials ................................................................................................ 63
5.2.3. Cell Culture Methods ............................................................................ 63
5.2.3.1. Plating and culturing cells ................................................................ 63
5.2.3.2. Lysing cells ......................................................................................... 64
5.2.3.3. Exposing cells to UV .......................................................................... 64
5.2.4. alamarBlue assay ................................................................................... 65
5.2.5. Superoxide dimutase (SOD) Activity................................................... 65
5.2.6. Caspase assay (Apoptotic Activity) ...................................................... 66
5.3. Results ............................................................................................................ 66
5.3.1. UV Exposure of 60mJ/cm2 resulted in response from the cells ........ 66
5.3.1.1. Western Blot for MMP-1 .................................................................. 67
5.3.1.2. Cell Viability assay ............................................................................ 68
5.3.1.3. Caspase Assay .................................................................................... 69