Polyethylenimine-derived gene carriers and their complexes with plasmid DNA [Elektronische Ressource] : design, synthesis and characterization / presented by Uta Lungwitz

Sujets

• Gesundheit

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 62
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Polyethylenimine-derived Gene
Carriers and their Complexes with
plasmid DNA

Design, Synthesis and Characterization

Dissertation to obtain the Degree of Doctor of Natural
Sciences (Dr. rer. nat.)
from the Faculty of Chemistry and Pharmacy
University of Regensburg



Presented by
Uta Lungwitz
From Greifswald

August 2006

To my family















This work was carried out from October 2001 until August 2006 at the
Department of Pharmaceutical Technology of the University of Regensburg.




The thesis was prepared under the supervision of Prof. Dr. Achim Göpferich










thSubmission of the Ph.D. application: July, 12 2006
thDate of examination: August, 10 2006

Examination board: Chairman: Prof. Dr. S. Elz
1. Expert: Prof. Dr. A. Göpferich
2. Expert: Prof. Dr. A. Buschauer
3. Examiner: Prof. Dr. J. Heilmann
Table of Contents

Chapter 1 Introduction: Polyethylenimine-based non-viral gene 7
delivery systems

Chapter 2 Goal of the thesis 55

Chapter 3 Synthesis of per-N-methylated polyethylenimine and its 61
application for non-viral gene delivery

Chapter 4 Synthesis and characterization of poly (2-ethyl-2- 85
oxazoline) and linear polyethylenimine

Chapter 5 Low molecular weight polyethylenimine-plasmid DNA 113
polyplexes: Particle properties and transfection efficacy

Chapter 6 Degradable low molecular weight linear 147
polyethylenimines for non-viral gene transfer

Chapter 7 Methoxy poly (ethylene glycol) – low molecular weight 181
linear polyethylenimine – derived copolymers enable
polyplex shielding

Chapter 8 Summary and conclusions 211

Appendix Abbreviations 219
Curriculum Vitae
List of publications
Acknoledgments




1. POLYETHYLENEIMINE-BASED NON-
VIRAL GENE DELIVERY SYSTEMS

U. Lungwitz, M. Breunig, T. Blunk, A. Göpferich



Department of Pharmaceutical Technology, University of Regensburg, D-
93053 Regensburg, Germany












European Journal of Pharmaceutics and Biopharmaceutics, 60 (2) 2005
p. 247-266 8 Introduction

Abstract
Gene therapy has become a promising strategy for the treatment of many
inheritable or acquired diseases that are currently considered incurable. Non-
viral vectors have attracted great interest, as they are simple to prepare,
rather stable, easy to modify and relatively safe, compared to viral vectors.
Unfortunately, they also suffer from lower transfection efficiency, requiring
additional effort for their optimization. The cationic polymer polyethylenimine
(PEI) has been widely used for non-viral transfection in vitro and in vivo and
has an advantage over other polycations in that it combines strong DNA
compaction capacity with an intrinsic endosomolytic activity. Here we give
some insight into strategies developed for PEI-based non-viral vectors to
overcome intracellular obstacles, including the improvement of methods for
polyplex preparation and the incorporation of endosomolytic agents or
nuclear localization signals. In recent years, PEI-based non-viral vectors
have been locally or systemically delivered, mostly to target gene delivery to
tumor tissue, the lung or liver. This requires strategies to efficiently shield
transfection polyplexes against non-specific interaction with blood
components, extracellular matrix and untargeted cells and the attachment of
targeting moieties, which allow for the directed gene delivery to the desired
cell or tissue. In this context, materials, facilitating the design of novel PEI-
based non-viral vectors are described. Introduction 9

Table of contents

1. Introduction........................................................................................................10
2. Intracellular pathway..........................................................................................10
3. Polyethylenimine (PEI)......................................................................................13
3.1. Branched PEI (bPEI) ................................................................................ 13
3.2. Linear PEI (lPEI)....................................................................................... 16
4. Endosomolysis..................................................................................................18
5. Nuclear targeting...............................................................................................21
6. Applications.......................................................................................................25
6.1. Polyplex shielding.....................................................................................27
6.1.1. PEGylation...........................................................................................
6.1.2. Pluronic................................................................................................30
6.1.3. Polyacrylic acid30
6.1.4. Poly (N-(2-hydroxypropyl)methacrylamide) [pHPMA]-derivatives ........ 30
6.1.5. Transferrin (Tf)......................................................................................33
6.2. Local application.......................................................................................33
6.2.1. Lung.....................................................................................................
6.2.2. Tumor...................................................................................................34
6.2.3. Brain34
6.3. Systemic administration and receptor targeting ....................................... 35
6.3.1. Glycosylated vehicles...........................................................................36
6.3.2. Transferrin receptor targeting............................................................... 38
6.3.3. Growth factors......................................................................................40
6.3.4. Membrane folate-binding protein.......................................................... 40
6.3.5. Integrins................................................................................................41
6.3.6. Antibodies and antibody fragments ...................................................... 42
7. Summary and outlook........................................................................................ 42
References............................................................................................................... 45 10 Introduction

1. Introduction
In recent years the knowledge of the molecular mechanisms of many
inheritable or acquired diseases has been greatly expanded, directing great
[1-6] [7]attention to the field of gene therapy . Antisense, ribozyme strategies or
[8]iRNA could potentially be used to downregulate or inactivate the
[9]expression of specific genes. In addition, suicide gene therapy could
enable the selective destruction of e.g. tumor cells using prodrug-converting
enzymes and tumor specific promoters.
Viral vectors have been applied to deliver therapeutic genes into living cells,
but their broad use is affected by the limited size of the genetic material that
[10;11]can be delivered and severe safety risks , based upon their
[12-14]immunogenicity and their oncogenic potential .
In light of these concerns, non-viral gene delivery has emerged as a
[15-18]promising alternative. Among the variety of different materials which
have been utilized in the manufacture of non-viral vectors, the use of
polymers confers several advantages, due to their ease of preparation,
purification and chemical modification as well as their enormous stability. The
polyamine PEI has emerged as a potent candidate, even though the use of
PEI-derived gene delivery vehicles is still limited by a relatively low
[19;20]transfection efficiency and short duration of gene expression compared
[21;22]to viral transfection systems, as well as cytotoxic effects .
In view of the great diversity within the field of non-viral gene delivery, here
we chose to focus on polyethylenimine-based transfection vehicles, including
strategies for their optimization and the observed effects thereof.
2. Intracellular pathway
Despite the broad experimental use of PEI-based vectors, the efficiency of
gene delivery remains insufficient. As understanding of the intracellular
trafficking pathways has expanded, the major hurdles of gene transfer at the