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  • Comparison of CT and Optical Image-based Assessment of Liposome Distribution

    by

    Huang Huang

    A thesis submitted in conformity with the requirements for the degree of Master of Science

    Graduate Department of Pharmaceutical Sciences University of Toronto

    © Copyright by Huang Huang 2012

  • ii

    Comparison of CT and Optical Image-based Assessment of

    Liposome Distribution

    Huang Huang

    Master of Science

    Department of Pharmaceutical Sciences

    University of Toronto

    2012

    Abstract

    The use of multimodal imaging as a tool to assess the in vivo pharmacokinetics and

    biodistribution of nanoparticles is important in drug development and imaging-guided therapy.

    The current study reports the use of combined computed tomography (micro-CT) and optical

    imaging to quantitatively assess the whole body (macro) and intratumoural (micro) distribution

    of a nano-sized liposome-based CT/optical imaging probe carrying iohexol and Cy5.5 over a

    study period of eight days. The liposomes were found to have a vascular half-life of 30.3 ± 8.9 h

    in mice bearing subcutaneous H520 non-small cell lung cancer (NSCLC) tumours with the

    maximum liposome accumulation in tumour achieved 48 h post injection. The liposome

    accumulation in tumour was quantitatively assessed using both micro-CT and fluorescence

    molecular tomography (FMT), where micro-CT was used to guide the FMT tumour delineation

    and signal correction. In situ confocal laser-scanning fluorescence microscopy analysis at the

    tumor site revealed that most of the liposomes remain in the vasculature at early time points (24

    h) with the majority having extravasated into the tumor interstitium at later time points (eight

    days). This investigation demonstrates the critical role micro-CT can play in guiding FMT-based

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    quantification of distribution. As well the combination of CT and optical imaging enable

    visualization of the liposomes at the whole body, tumor and cellular levels with high sensitivity

    and excellent anatomical background. Such non-invasive assessment of therapeutic distribution

    at the macro and micro scale is necessary for implementation of personalized medicine including

    image-guided patient stratification and real-time adjustment of therapeutic dose.

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    Acknowledgments

    It is a great pleasure to thank the many people who made this thesis possible.

    Foremost, I would like to express my sincere gratitude to my supervisor, Dr. Christine Allen.

    With her enthusiasm, her inspiration, and her continuous support for my study and research, she

    helped me enjoy the research I did. I deeply appreciate all her contributions of time and patience

    to make my M.Sc. experience exciting. Through my thesis writing, she provided encouragement,

    sound advice, and good teaching. It has been a pleasure to be her student.

    My committee member and unofficial co-supervisor, Dr. David Jaffray, has provided a lot of

    guidance to me during this research. I am especially grateful for his unique thinking and diverse

    knowledge, which provided me many ideas that have been very helpful. I appreciate Drs. Allen

    and Jaffray for obtaining the funding that has supported my research.

    I would like to thank my other committee member, Dr. Gang Zheng, for taking his time to

    critically review my work and offer constructive suggestions.

    My sincere thanks to all my colleagues and friends in the Allen lab and at STTARR, past and

    present. In particular, I would like to express my special gratitude toward John Lo and Michael

    Dunne. John took the time to train me as an undergraduate student, and provided collaborative

    help that paved the way to this research. I thank Michael for using the H520 tumour model he

    developed in our lab. His generous help on tumour cell preparation and animal inoculation

    described in this research is also deeply appreciated. I would also like to thank Dr. Jinzi Zheng

    for providing scientific support, and Debbie Squires for technical assistance with the

    administration of the contrast agents.

    Finally, I would like to thank my beloved family and friends for all their love, support and

    encouragement throughout my studies. Thank you all.

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    Table of Contents

    Acknowledgments.......................................................................................................................... iv

    Table of Contents............................................................................................................................ v

    List of Tables ................................................................................................................................ vii

    List of Figures .............................................................................................................................. viii

    List of Appendices ......................................................................................................................... xi

    CHAPTER 1 GENERAL INTRODUCTION .............................................................................. 1

    1.1 Goal, Objectives and Rationale........................................................................................... 1

    1.2 Liposomes ........................................................................................................................... 3

    2.2.1 Physico-chemical Properties................................................................................... 3

    2.2.2 Liposomes as Drug Carriers.................................................................................... 6

    1.3 Multimodal Imaging ......................................................................................................... 10

    2.2.1 CT Imaging ........................................................................................................... 11

    2.2.2 Optical Imaging .................................................................................................... 12

    2.2.3 CT/Optical Multimodal Imaging .......................................................................... 14

    1.4 Image-based Assessment of Nanoparticle Distribution.................................................... 15

    CHAPTER 2 Comparison of CT and Optical Image-based Assessment of Liposome Distribution .............................................................................................................................. 19

    2.1 Introduction....................................................................................................................... 20

    2.2 Materials and Methods...................................................................................................... 23

    2.2.1 Synthesis of Cy5.5-DSPE ..................................................................................... 23

    2.2.2 Liposome Preparation ........................................................................................... 24

    2.2.3 Liposome Characterization ................................................................................... 24

    2.2.4 In vitro Stability and Agent Release ..................................................................... 25

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    2.2.5 In vivo CT/Optical Imaging .................................................................................. 26

    2.2.6 Image Analysis...................................................................................................... 27

    2.3 Results............................................................................................................................... 30

    2.3.1 Synthesis and Characterization of Cy5.5-DSPE................................................... 30

    2.3.2 Physicochemical Characterization of the Liposomes ........................................... 31

    2.3.3 In vivo Pharmacokinetics, Biodistribution of the Liposomes ............................... 35

    2.4 Discussion ......................................................................................................................... 46

    CHAPTER 3 Conclusion............................................................................................................. 50

    3.1 Conclusions....................................................................................................................... 50

    3.2 Future Directions .............................................................................................................. 52

    References..................................................................................................................................... 54

    Appendix....................................................................................................................................... 67

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    List of Tables

    Table 2.1 Physicochemical characteristics of CT and CT/optical liposomes (n=5). Data are represented as the mean ± standard deviation .............................................................. 32

    Table 2.2 Pharmacokinetic parameters for CT and CT/optical liposomes (n=7) in non- tumour-bearing mice and CT/optical liposomes (n=5) in tumour-bearing mice. St