Handbook of Nonmedical Applications of Liposomes

From Gene Delivery and Diagnosis to Ecology

Danilo D. Lasic, Yechezkel Barenholz

• Publisher: CRC Press
• Year: 2019
• Print ISBN: 9780367261023
• eBook ISBN: 9781000697636
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Biochemistry in Medicine
• Index: Biological Sciences

First published in 1996, liposomes have become an important model in fundamental biomembrane research, including biophysical, biochemical, and cell biological studies of membranes and cell function. They are thoroughly studied in several applications, such as drug delivery systems in medical applications and as controlled release systems, microencapsulating media, signal carriers, support matrices, and solubilizers in other applications. While medical applications have been extensively reviewed in recent literature, there is a need for easily accessible information on applications for liposomes beyond pharmacology and medicine. The Handbook of Nonmedical Applications of Liposomes fills this void.This unique new handbook series presents recent developments in the use of liposomes in many scientific disciplines, from studies on the origin of life, protein function, and vesicle shapes, to applications in cosmetics, diagnostics, ecology, bioreclamation, and the food industry. In these volumes many of the top experts contribute extensive reviews of their work.

Chapter 1

Liposomes in Gene Therapy

Danilo D. Lasic

CONTENTS

• I. Introduction
• II. Gene Therapy
• III. Historical Perspective
• IV. Cationic Liposomes
• V. Transfection and Gene Expression
• VI. Mechanism of Transfection
• References

I. Introduction

Gene therapy and diagnostics may become the major development in medicine in the next century/millennium due to extremely rapid developments in recombinant DNA technology, a greater understanding of genetic and some acquired diseases on the molecular level and mapping of the human genome, as well as improved ways to deliver recombinant genetic codes into appropriate cells.

II. Gene Therapy

Several thousand diseases can be linked to defective or missing genes and the concept is that by bringing the appropriate gene into appropriate cells, the mutated or missing proteins can be synthesized and alleviate the signs of the disease. Furthermore, in some infectious diseases, inflammation states, or in cancer, one can stimulate the immune system to produce and secrete more cytokines (such as various colony stimulating factors and tumor necrosis factor, interleukins, interferons) and via enhanced cytotoxicity and/or enhanced number of killer cells, exterminate the malignancy. In addition to a “turn on” concept as described above (and in which gene therapy aims at replacing nonfunctional gene and/or adding its functional copy), there is also a therapy possible by switching genes off. This includes the so-called antisense (oligonucleotides) technology whose aim is to deliver a short, normally single strand oligonucleotide with a complementary sequence, to the part of the unwanted gene or messenger RNA, or to form a triple helix with DNA, and to stop the process, such as cancer, scar tissue, or other undesired cell growth. Gene therapy in a broader sense encompasses also enhancement of the immune system, tagging neoplastic cells for autoimmune destruction and vaccination in which the body itself produces antigens to induce an immune response. The aim of the treatment is to transfect cells with genes reconstituted into plasmids without incorporating genetic material into chromosomes — in order to avoid possible side effects such as cancer or viral infection. This approach therefore requires either rather frequent dosing or development of self-replicating plasmids which remain in the cell nucleus during cell division. Alternatively, integration of DNA can be accomplished by site-specific integration of cDNA flanked by specific DNA sequences, homologous recombination of genomic DNA, or random integration.

It seems that functional plasmids containing powerful gene expression enhancers and with cell specific promoters can be now routinely constructed, while the delivery, (not only in vitro and ex vivo, but especially in vivo), presents the largest challenge. Currently, in most therapies in humans, appropriate cells are taken out and after transfection in a Petri dish, injected back into the patient. Obviously, the goal of these treatments is in vivo administration of appropriate plasmids or their constructs with various carriers. The targeted diseases are cancer, cardiovascular diseases, cystic fibrosis, and many others.

Large size and charge of plasmids (3-15 kilobase pairs = 2-10 million Da = 6-30 thousand negative charges with linear length of molecule 1-5 μm) make the intracellular delivery of plasmids extremely difficult. In addition, free DNA is quickly degraded when applied systemically. Ex vivo approaches can use physical and chemical transfection methods such as electroporation, direct injection, and calcium precipitation. Delivery systems include also gene gun and precipitation with some polymers and differ mostly with respect to transfection efficacy, safety, plasmid loading capacity, immunogenicity, and nature of their interactions with cells. The most frequently used delivery systems in vivo are viral constructs, i.e., genes inserted into viral genes and reconstituted into noninfective viruses. Another approach involves complexation of the plasmid with liposomes, (cationic) polymers and oligomers, polyvalent cations, poly cations, or any combination of these approaches. Naked DNA can be also injected locally. In addition to intravenous administration, direct intramuscular, subcutaneous, localized, and intraperitoneal injection, and instillation into airways as well as inhalation of an aerosol of such particles, can be used.

DNA is inserted either in a viral vector or in a bacterial plasmid which can thus be introduced into cells either via viral or internalizing (endocytosis, fusion) pathway. The most widely used are retroviruses. They are single-strand RNA viruses which incorporate the gene directly into chromosome of the target cell. This presents a safety concern due to a potential carcinogenicity and infectivity despite the fact that several viral protein codes are deleted. Also, contamination with intact retroviruses, which can be highly oncogenic, cannot be ruled out. Adenoviruses are less dangerous (an example is a common cold virus). They are double-stranded viruses which can incorporate larger genes than retroviruses. They do not incorporate into the genome and this reduces the risk of malignant transformations but also reduces the persistence of expression. Also, an immune response against viral proteins may develop, besides the fact that many people possess an immunity against these viruses. Other viruses, such as adeno-associated viruses or herpes simplex virus, are also employed.^1,2

Because of some inherent problems associated with viral vectors, such as potential carcinogenicity, infectivity, and development of an immune response, many researchers believe that liposome- or lipid-based transfection, possibly in conjunction with some other DNA condensing agent, will become the mainstream of gene therapy.

III. Historical Perspective

The first attempts to encapsulate and transfect DNA using liposomes were undertaken in the late 1970s.³ However, despite some successes, the protocols were cumbersome and practically inapplicable in vivo, and with the introduction of electroporation these approaches were largely phased out.^4,5

Following successful DNA complexation with cationic polymers, cationic lipids were used to coat DNA. This lipid coating was followed by complexation with cationic liposomes and several cationic lipids were introduced.^{6,7,8,9,10,11,12,13,14} A variety of different cationic lipids was synthesized which showed great differences in transfection activity. With the exception of positively charged cholestérols and some variation on DOTMA,^9,10,11,12 at present, however, no structure-activity relationships are known. Another goal of lipid synthesis is also to produce the safest cationic compounds. For that reason it is desirable that they contain biodegradable chemical bonds. It was realized that in order to improve transfection efficacy, liposomes have to contain also a neutral lipid. Most of the studies report that cationic lipid plus dioleoyl phosphatidyl ethanolamine (DOPE) at approximately 50 mol% yields the highest transfection efficiency. This effect was explained because of its ability to form a hexagonal II phase and to facilitate either complexation or internalization of the DNA-liposome complex (genosomes) into cells and perhaps, facilitate the release of the complex/DNA from the endosome after endocytosis.^9,10

IV. Cationic Liposomes

Even more than the influence of neutral lipid, the nature of cationic lipid and its structure-activity relation were studied.^9,11,12 Various formulations, containing lipids such as DODAB, DOTAP, DOTMA, DOGS, DOSPA, and DC-Choi (formulae are shown in the following papers), resulted in several commercial transfection formulations which showed rather good transfection efficacies in various cell models but were practically ineffective for in vivo applications.

In the first 20 years of liposome research, cationic liposomes were not studied extensively due to their toxicity. Colloidal studies have shown that DLVO theory can be applied to explain their stability, although in the case of s...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• General Preface
• Introduction to Volume IV Liposomes: from Gene Delivery and Diagnostics to Ecology
• The Editors
• Contributors
• Contents
• Chapter 1 Liposomes in Gene Therapy
• Chapter 2 Structural Features of DNA-Cationic Liposome Complexes and Their Implication for Transfection
• Chapter 3 Delivery of DNA, RNA, and Proteins by Cationic Liposomes
• Chapter 4 Advances in the Design and Application of Cytofectin Formulations
• Chapter 5 Gene Transfer to Plants by Lipofection and Electroporation
• Chapter 6 The Contribution of Cationic Derivatives of Phospholipids and Cholesterol to the Bioadhesiveness of Liposomes and Their Transfection Performance
• Chapter 7 Liposomes in Cosmetics: How and Why?
• Chapter 8 Transport of Liposome-Entrapped Molecules into the Skin as Studied by Electron Paramagnetic Resonance Imaging Methods
• Chapter 9 Some Large-Scale, Nonmedical Applications of Nonphospholipid Liposomes
• Chapter 10 Liposomal Formulations of Agrichemical Pesticides
• Chapter 11 Liposomes and Cheesemaking
• Chapter 12 Use of Liposomes for Wool Dyeing
• Chapter 13 Liposomes as a Model System to Study Shark Repellency
• Chapter 14 Use of Antigen-Coupled Liposomes for Homogeneous Immunoassays of Polyclonal Antibody
• Chapter 15 Liposomes in Immunodiagnostics
• Chapter 16 Stehe Stabilization of Liposomes Improves Their Use in Diagnostics
• Chapter 17 Liposome-Amplified Immunodetermination of Environmental Contaminants
• Chapter 18 The Use of Liposomes in Biodegradability Testing
• Chapter 19 Microbe Entrapment in Giant Liposomes
• Chapter 20 Liposomes as a Model for Membrane Structures and Structural Transformations: A Liposome Album
• Chapter 21 Liposomes: Past, Present, and Future
• Index