Liposomes

Liposomes were first described in 1965 as a model of cellular membranes and quickly were applied to the delivery of substances to cells. Liposomes entrap DNA by one of two mechanisms which has resulted in their classification as either cationic liposomes or pH-sensitive liposomes.

Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. Cationic liposomes consist of a positively charged lipid and a co-lipid. Commonly used co-lipids include dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC). Co-lipids, also called helper lipids, are in most cases required for stabilization of liposome complex. A variety of positively charged lipid formulations are commercially available and many other are under development. One of the most frequently cited cationic lipids is lipofectin. Lipofectin is a commercially available cationic lipid first reported by Phil Felgner in 1987 to deliver genes to cells in culture. Lipofectin is a mixture of N-[1-(2, 3-dioleyloyx) propyl]-N-N-N-trimethyl ammonia chloride (DOTMA) and DOPE. The structure of DOTMA is shown below.

DNA and lipofectin interact spontaneously to form complexes that have a 100% loading efficiency. In other words, all of the DNA is complexed with the lipofectin, provided enough lipofectin is available. It is assumed that the negative charge of the DNA molecule interacts with the positively charged groups of the DOTMA. The lipid:DNA ratio and overall lipid concentrations used in forming these complexes are extremely important for efficient gene transfer and vary with application. Lipofectin has been used to deliver linear DNA, plasmid DNA, and RNA to a variety of cells in culture. Shortly after its introduction, it was shown that lipofectin could be used to deliver genes in vivo. Following intravenous administration of lipofectin-DNA complexes, both the lung and liver showed marked affinity for uptake of these complexes and transgene expression. Injection of these complexes into other tissues has had varying results and, for the most part, are much less efficient than lipofectin-mediated gene transfer into either the lung or the liver.

pH-sensitive, or negatively-charged liposomes, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Yet, some DNA does manage to get entrapped within the aqueous interior of these liposomes. In some cases, these liposomes are destabilized by low pH and hence the term pH- sensitive. To date, cationic liposomes have been much more efficient at gene delivery both in vivo and in vitro than pH-sensitive liposomes. pH-sensitive liposomes have the potential to be much more efficient at in vivo DNA delivery than their cationic counterparts and should be able to do so with reduced toxicity and interference from serum protein.

Applications. Liposomes offer several advantages in delivering genes to cells. (1) Liposomes can complex both with negatively and positively charged molecules. (2) Liposomes offer a degree of protection to the DNA from degradative processes. (3) Liposomes can carry large pieces of DNA, potentially as large as a chromosome. (4) Liposomes can be targeted to specific cells or tissues. In addition, liposomes overcome problems inherent with viral vectors - specifically concerns of immunogenicity and replication competent virus contamination. The ability to chemically synthesize a wide variety of liposomes has resulted in a highly adaptable and flexible system capable of gene delivery both in vitro and in vivo. As liposome technology is better understood, it should be possible to produce reagents with improved in vivo gene delivery into specific tissues. Studies involving protein-DNA-liposome complexes are already showing promise in the ability to target DNA delivery into specific cells. Current limitations regarding in vivo application of liposomes revolve around the low transfection efficiencies and transient gene expression. Also, liposomes display a small degree of cellular toxicity and appear to be inhibited by serum components. The ability to overcome these problems should greatly facilitate their application to a variety of gene delivery mechanisms.　

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