WWW.DISSERTATION.XLIBX.INFO
FREE ELECTRONIC LIBRARY - Dissertations, online materials
 
<< HOME
CONTACTS



Pages:   || 2 | 3 | 4 | 5 |   ...   | 9 |

«(肝臓類洞内皮細胞へのsiRNA送達を目的とした革新的な能動的標 的化システムの構築) A Dissertation Submitted To The ...»

-- [ Page 1 ] --

Instructions for use

Development of a novel active targeting system for siRNA

delivery to liver sinusoidal endothelial cell in vivo

(肝臓類洞内皮細胞へのsiRNA送達を目的とした革新的な能動的標

的化システムの構築)

A Dissertation

Submitted To

The Graduate School of Life Science

Hokkaido University

In Partial Fulfillment of the Requirements

For The Degree of

Doctor of Philosophy

Laboratory of Innovative Nanomedicine

Department of Biomedical and Pharmaceutical Science Graduate School of Life Science Hokkaido University Hokkaido, Sapporo, Japan Afsana Akhter September 2013 Development of a novel active targeting system for siRNA delivery to liver sinusoidal endothelial cell in vivo A Dissertation Submitted by Afsana Akhter In partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Pharmaceutical Sciences (Faculty of Pharmaceutical Sciences) Hokkaido University Hokkaido, Sapporo, Japan 2013 Supervisor Prof. Dr. Hideyoshi Harashima Review Committee Prof. Dr. Mitsuru Sugawara Associate Prof. Dr. Yoh Takekuma Associate Prof. Dr. Hidetaka Akita Dedication To my beloved Parents Acknowledgements First I would like to express my deepest thanks to my supervisor Prof. Hideyoshi Harashima for accepting me as a graduate student in his laboratory. I am also indebted to him for providing me the opportunity to complete my PhD thesis. He has been so supportive since the days I began working. Ever since, his patience, motivation, enthusiasm, and immense knowledge always inspired me to continue my study. He has been actively interested in my work and has always been available to advise me.

Thanks to him for giving me the opportunity to conduct my research career in the field of Nanotechnology. His guidance will be the key to take the challenges as well as to build up my future career.

This work would not have been possible without the encouragement and guidance of Dr.

Yasuhiro Hayashi, former member of this lab. Words are too short to represent his countless efforts to teach me the basic things that I required for my research, generate new ideas and to get good results. Lot more thanks to him for intellectual brilliance and encouragement. Since the first day of our introduction, to all the way towards my research, he provided me the support I needed at every step. It is his careful supervision that brought me wherever I stand today in my research career. Thank you for your kind assistance and insight on completing this paper.

I also want to express my deep gratitude to Dr. Yu Sakurai for his continuous suggestion not only in writing this thesis paper but also in experimental works. I am also expressing my appreciation to Dr. Hidetaka Akita, Dr. Kazuaki Kajimoto, Dr.

Yuma Yamada, Dr. Hiroto Hatakeyama, Dr. Mamoru Hyodo, Dr. Takashi Nakamura, for their advice during my study.

I am grateful to Dr. Kyoko Hida and Dr. Noritaka Ohga, Division of Vascular Biology, Graduate School of Dental Medicine, Hokkaido University for their kind assistance;

especially for the liver endothelial cells that I used in my research.

My sincere thanks also go to the members of my doctoral committee for their input, valuable discussions and suggestions. I am expressing my gratitude to Prof. Mitsuru Sugawara, Laboratory of Pharmacokinetics, Hokkaido University, who was the Chairman in my doctoral thesis presentation. I am also thankful to the reviewers of my thesis paper for their kind observations and expert opinions.

I want to thank present and past members of the lab: Chisato Nagai, Jun Yamauchi, Mina Tamaru, Naoyuki Toriyabe, Yuki Noguchi, Erina Suemitsu for giving me the introductory lessons about the lab and for sharing their knowledge.

I am also expressing my gratefulness to the Staff of Faculty of Pharmaceutical Sciences, Hokkaido University for their response to me; whenever I go to them they always welcome and support me. Very special thanks to Eiko Abe who made all the administrative issues easier for my stay in Japan.

My deepest gratitude goes to my family for their continuous love and support throughout my life; this thesis is simply impossible without them. I am indebted to my father, Muhammad Nasir and mother, Raihana Begum; for the love and support they have provided throughout my entire life. They have never complained in spite of all the hardships in their life. I am ever grateful to their unselfish blessings and supports. I also want to thank my brother Rakibul Hasan and my in-law parents for their faith and mental supports.

Finally, and most importantly, I would like to thank my husband Dr. Golam Kibria for his untiring support, encouragement, patience and love, which strengthen me to go ahead to my future dream. I am expressing my deepest love and affection to my son Tajwar Morshed (Ayaan), whose presence in my life strengthened me to take every challenges of life. This work would not come true without their sacrifices.

Above all I want to give thanks the omnipresent Allah, for answering my prayers for giving me the strength to finish this thesis work in time.

TABLE OF CONTENTS

–  –  –

1. General Introduction

1.1. Liver …………………..………………………………





1.2. Structure of liver……………………………………………………..…………… 2

1.3. Different types of cells in liver and their function…………………..…………3

1.4. Biological features of Liver sinusoidal endothelial cell (LSEC)………….….4

1.5. General functions of LSEC…………………………………………………………………..5

1.6. Phenotype of LSEC……………………………………………………………………….......5

1.7. Morphology of LSEC………………………………………………………………………….7 1.7.1 Fenestration……………………………………………………………………….7 1.7. 2. Contraction and dilatation mechanism of fenestrae……………………….7

1.8. Liver Diseases and LSEC…………………………………………………………………..9 1.8.1. Hepatitis………………………………………………………………………….9 1.8.2. Cirrhosis and Fibrosis………………………………………………………….10 1.8.3. Sinusoidal Obstruction Syndrome (SOS)……………………………………10 1.8.4. Radiation-Induced Liver Disease (RILD)……………………………………11 1.8.5. Alcoholic Liver diseases (ALD)………………………………………………..11

1.9. Pathophysiology of liver diseases…………………………………………………………12 1.9.1. Inflammation and other liver injury…………………………………………12 1.9.2. Chemokines and cytokines……………………………………………………13

1.10. Role of different types of cells in liver inflammation………………………………….14

–  –  –

1.10.1. Role of hepatocytes…………………………………………………………....14 1.10.2. Role of Sinusoidal endothelial cells…………………………………………15 1.10.3. Role of hepatic stellate cells………………………………………………….17 1.10.4. Role of Kupffer cells…………………………………………………………...17

1.11. siRNA delivery to Liver……………………………………………………………………17 1.11.1. Advantages of siRNA delivery……………………………………………….18 1.11.2. Challenges in siRNA delivery……………………………………………….18 1.11.3. Recent approaches in the delivery of siRNA to liver……………………18 1.11.4. Liver sinusoidal endothelial cell targeted siRNA delivery…………….20

2. Development of LSEC selective ligand modified liposomes

2.1. Introduction…………………………………………………………………………………..21

2.2. Materials and methods…………………………………………………………..…………23 2.2.1. Materials………………………………………………………………..………..23 2.2.2. Animals…………………………………………………………………………...23 2.2.3. Conjugation of the RLTR peptide to PEG2000-DSPE………..……………..23 2.2.4. Preparation of liposomes……………………………………………………....24 2.2.5. Isolation of Primary liver endothelial cells (LSECs)………………………24 2.2.6. In vitro Cellular uptake study……………………………….………………..24 2.2.7. In vivo Biodistribution study………………………………………………….25 2.2.8. Confocal microscopy experiment………………………………………….…..25 2.2.9. Inhibition assay……………………………………………………………….…26 2.2.9.1 In vivo competitive inhibition study of RLTR-PEG-LPs…………………26 2.2.9.2 In vitro competitive inhibition study of RLTR-PEG-LPs…...…...………26 2.2.10. Statistical analysis…………………………………………………………….26

2.3. Results…………………………………………………………………………….................26 2.3.1. Synthesis of RLTR-PEG-DSPE……………………………………………….26 2.3.2. The characteristic of RLTR-PEG-LPs and its cellular uptake…………...28 2.3.3. In vivo selectivity of RLTR peptide…………………………………………..28 2.3.4. In vivo inhibition study………………………………………………………...3

–  –  –

Formulation of new peptide-conjugated MEND and delivering siRNA to LSEC

3.1. Introduction……………………………………………………………

3.2. Materials and methods………………………………………………………….………….40 3.2.1. Materials………………………………………………………………….………….40 3.2.2. Experimental animals…………………………………………………….….……41 3.2.3. Cell culture……………………………………………………………….….………41 3.2.4. Conjugation of the KLGR peptide to PEG2000-DSPE……………..….………..41 3.2.5. Preparation of MEND…………………………………………………………..….41 3.2.6. Ribogreen assay………………………………………………………………….…42 3.2.7. In vivo gene delivery study…………………………………….…………….……42 3.2.8. RNA extraction and PCR analysis……………………………….…….………42 3.2.9. Confocal microscopy experiment…………………………………………………43 3.2.10. In vitro quantitative cellular uptake study……………………………….…..43 3.2.11. Endosomal escape efficiency of STR-KLGR modified MEND………….…..44 3.2.12. Uptake mechanism study……………………………………………………..…44 3.2.13.Toxicological study………………………………………………………………...45 3.2.14. Statistical analysis………………………………………………………………..45

3.3. Results………………………………………………………………………………………...45 3.3.1. Synthesis of KLGR-PEG-DSPE……………………………………………….….45 3.3.2. The characteristic of KLGR-PEG or STR-KLGR modified YSK05 MEND...46 3.3.3. In vivo gene silencing activity of KLGR-PEG or STR-KLGR modified YSK05 MEND……………………………………………………………………….…….…47 3.3.4. In vivo accumulation of STR-KLGR modified YSK05-MEND in liver……...48 3.3.5. Cellular uptake by flow cytometry…………...…………………………..………49 3.3.6. Effect of low density of STR-KLGR at YSK05-MEND in in vivo gene silencing activity……..……………………………………………………….……50

–  –  –

3.4. Discussion……………………………………………………………...……………………..60

3.5. Conclusion……………………………………………………………………..……………..64 Summary of the study……………………………………………………..……………65 References………………………………………………………………………….……….....67 List of Figures…………………………………………………………………………….....78 List of Tables…………………………………………………………………………………81 Appendix Structure of lipids and peptides used in this study…………………………………………..vi Abbreviations…………………………………………………………………………………….viii

iv Aim of the study.

Liver sinusoidal endothelial cell (LSEC) has become an interest of study for some recent years as it plays some important role in both physiological and pathophysiological condition. On the basis of this consideration the main aim of this study was to develop a peptide modified MEND that can be extended for systemic in vivo application for intracellular delivery of small-interfering RNA (siRNA) to LSEC. The different specific

steps on the way to achieve this aim are the following:



Pages:   || 2 | 3 | 4 | 5 |   ...   | 9 |


Similar works:

«Isolated Experiences: Gilles Deleuze and the Solitudes of Reversed Platonism James Brusseau Facultad de Filosofia y Letras Universidad Nacional Autonoma de Mexico Acknowledgement I acknowledge Professor Alphonso Lingis for his contributions to this work. The task of contemporary philosophy has been defined: the reversal of Platonism.Gilles Deleuze, 1968 Difference and Repetition Contents Introduction I Difference 1. Difference As Production And Limitation. 2. The Eternal Return Does Difference:...»

«DEVOLUTION FROM ABOVE: THE ORIGINS AND PERSISTENCE OF STATE-SPONSORED MILITIAS A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Government By Ariel I. Ahram, M.A. Washington, DC July 30, 2008 Copyright 2008 by Ariel I. Ahram All Rights Reserved ii DEVOLUTION FROM ABOVE: THE ORIGINS AND PERSISTENCE OF STATE-SPONSORED MILITIAS Ariel I. Ahram, M.A. Thesis...»

«Humanity at the Turning Point: Rethinking Nature, Culture and Freedom (Sonja Servomaa, editor). Helsinki, Finland: Renvall Institute for Area and Cultural Studies, 2006.Beyond Tolerance: Globalization, Freedom, and Religious Pluralism Douglas W. Shrader1 Distinguished Teaching Professor & Chair of Philosophy SUNY Oneonta Oneonta, NY Abstract: If “Globalization” is to mean something other than imposing a single set of uniform, unexamined, and unchallengeable ideas on the entire human race,...»

«Stony Brook University The official electronic file of this thesis or dissertation is maintained by the University Libraries on behalf of The Graduate School at Stony Brook University. © Allll Riightts Reserved by Autthor. © A R gh s Reserved by Au hor Ecogeographic Variation in Neandertal Dietary Habits: Evidence from Microwear Texture Analysis A Dissertation Presented by Sireen El Zaatari to The Graduate School in Patrial fulfillment of the Requirements for the Degree of Doctor of...»

«COMPOSITE THERMAL CAPACITORS FOR TRANSIENT THERMAL MANAGEMENT OF MULTICORE MICROPROCESSORS A Dissertation Presented to The Academic Faculty by Craig Elkton Green In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the George W Woodruff School of Mechanical Engineering Georgia Institute of Technology August 2012 COMPOSITE THERMAL CAPACITORS FOR TRANSIENT THERMAL MANAGEMENT OF MULTICORE MICROPROCESSORS Approved by: Dr. Andrei G Fedorov, Advisor Dr. Yogendra K. Joshi,...»

«High-Speed Soft-Processor Architecture for FPGA Overlays by Charles Eric LaForest A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Electrical and Computer Engineering University of Toronto c Copyright 2015 by Charles Eric LaForest Abstract High-Speed Soft-Processor Architecture for FPGA Overlays Charles Eric LaForest Doctor of Philosophy Graduate Department of Electrical and Computer Engineering University of Toronto 2015...»

«Characterization, diagnosis and environmental survival of turkey arthritis reoviruses A DISSERTATION SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Sunil Kumar IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisors: Drs. Sagar M. Goyal and Robert E. Porter June 2014 © Sunil Kumar 2014 Acknowledgements My graduate experience has been like no other endeavor I have ever pursued. It has been a time of great growth, life changes, and learning. I am so...»

«Der Sechste Zhva dmar pa Chos kyi dbang phyug (1584–1630) und sein Reisebericht aus den Jahren 1629/1630: Studie, Edition und Übersetzung Inaugural-Dissertation zur Erlangung des Doktorgrades der Philosophie an der Ludwig-Maximilians-Universität München vorgelegt von: Navina Lamminger Datum der mündlichen Prüfung: 28. Januar 2013 Gutachter: Prof. Dr. Franz-Karl Ehrhard Prof. Dr. Jens-Uwe Hartmann Inhaltsverzeichnis Danksagung vii 1. Allgemeine Einführung und Aufbau der Arbeit 1 1.1...»

«EXPERIMENTAL CHARACTERIZATION AND MULTIDISCIPLINARY CONCEPTUAL DESIGN OPTIMIZATION OF A BENDABLE LOAD STIFFENED UNMANNED AIR VEHICLE WING By VIJAY NARAYAN JAGDALE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1 © 2010 Vijay Narayan Jagdale 2 To my idol and inspiring father, Late Narayan L Jagdale; my caring mother, Kaushalya N. Jagdale; my loving wife,...»

«FABRICATION, CHARACTERIZATION, AND APPLICATION OF MULTIFUNCTIONAL MICROCANTILEVER HEATERS A Dissertation Presented to The Academic Faculty by Jungchul Lee In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the George W. Woodruff School of Mechanical Engineering Georgia Institute of Technology May 2007 FABRICATION, CHARACTERIZATION, AND APPLICATION OF MULTIFUNCTIONAL MICROCANTILEVER HEATERS Approved by: Dr. William P. King, Advisor Dr. Mark G. Allen George W....»

«Jackie Feldman April, 2013 CURRICULUM VITAE AND LIST OF PUBLICATIONS Personal Details Jackie Feldman Born: August 31, 1956, New York, N.Y. Date of immigration: October 1978. Department of Sociology and Anthropology Ben Gurion University Beersheba, Israel Tel.: 08-647-2083 jfeldman@bgu.ac.il Home address: Anusei Mashhad 3, Apt. 20 Jerusalem 93784 Israel Tel. 052-8704489 Education B.A. 1973-77: City College of New York – Departments of Mathematics and Philosophy, magna cum laude. M.A. 1978-88:...»

«THE DEVELOPMENT OF THE TONER DENSITY SENSOR FOR CLOSED-LOOP FEEDBACK LASER PRINTER CALIBRATION by Keisha Josephine Thomas A dissertation submitted to the faculty of The University of North Carolina at Charlotte in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Electrical Engineering Charlotte 2009 Approved by: Dr. Ivan Howitt Dr. Tom Weldon Dr. Yogendra Kakad Dr. Andrew Willis Dr. Xintao Wu ii © 2009 Keisha Josephine Thomas ALL RIGHTS RESERVED iii...»





 
<<  HOME   |    CONTACTS
2016 www.dissertation.xlibx.info - Dissertations, online materials

Materials of this site are available for review, all rights belong to their respective owners.
If you do not agree with the fact that your material is placed on this site, please, email us, we will within 1-2 business days delete him.