DSouza - Liposomes: methods and protocols

Department of Pharmaceutical Sciences, Midwestern University College of Pharmacy Glendale, 19555 North 59th Ave., Glendale, AZ 85308, USA

Volkmar Weissig

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Following the first observations of living cells under a simple light microscope by Anton van Leeuwenhoek in the late 1600s, the question ultimately arose as to what holds it all together, i.e., what actually surrounds such a microscopic tiny structure. The history of trying to understand biological membranes [].

Among those landmarks of biomembrane history, a serendipitous observation made by Alex Bangham during the early 1960s deserves undoubtedly a special place. In the early 1960s, biological membranes were increasingly being visualized through the use of electron microscopy. Following appropriate staining, membranes showed up in electron micrographs as two dark bands separated by a light region [].

To prove this self-assembly behavior of hydrated phospholipids, however, Bangham and Horne had to work two more years and at no time did (they) imagine that (their) model system would prove to be anything like as useful as the BLM [].

In the following years, Alec Bangham kept demonstrating the usefulness of liposomes as a membrane model for studying fundamental membrane properties such as permeation but also adhesion and fusion [] have been investigated. The list could go on.

In addition to utilizing them as membrane models, Alec Bangham extended the use of liposomes to the study of the mechanism of action of drugs such as local anesthetics [].

One year before his death, in his very last publication at the age of 88 as the sole author (!), Alec Bangham sums up the impact of his serendipitous observation from the beginning of the 1960s on his scientific career by writing it was the odd pattern of a well-drawn drop of blood that initiated my curiosity and eased my career away from morbid anatomy to that of the physical chemistry of cell surfaces [] (reprinted with permission from Elsevier):

Based on their initial work about liposomal encapsulation of amyloglucosidase, Gregory Gregoriadis together with the Madam of London, i.e., Brenda Ryman proposed in 1971 liposomes as carriers of enzymes or drugs as a new approach to the treatment of storage diseases []. The type of liposomes as made by Gregoriadis and Ryman via probe sonication of a lipid suspension was years later named Small Unilamellar Vesicles (SUVs), which have become known for their general small encapsulation efficiency.

Back to 1971, Gregoriadis and Ryman injected the amyloglucosidase and labeled albumin-containing liposomes intravenously into rats. About 60 % of all liposomes were eliminated from the circulation within 10 min. Remarkably, using [3H]-cholesterol-labeled and 131I-albumin-loaded liposomes they found that circulating liposomes seemed to stay intact; no measurable leakage of 131I-albumin was found. Most of the radioactivity was recovered in liver and to some extent in the spleen, with a maximum of 56 % of the injected dose after the first 15 min. This rapid clearance of i.v. injected liposomes via organs of the RES system, as firstly described by Gregoriadis and Ryman in 1971, proved to become the major and almost insurmountable hurdle for getting liposome-based drugs into the clinic. Subsequently, during the middle and late 1980s the extreme short half-life of liposomes in circulation contributed to a general waning of the original enthusiasm associated with liposomes as a potentially universal drug carrier. This barrier toward the clinical application of liposomes fell in 1990, when Alexander Klibanov, who obtained his Ph.D. degree in Vladimir Torchilins lab in Moscow and at that time, worked as a Postdoc in Leaf Huangs lab then in Nashville, Tennessee grafted polyethylene glycol (PEG) chains onto the surface of liposomes in order to significantly prolong the circulation time of liposomes [].

Two years later, in a landmark paper in Nature [].

In the late 1990s, Gregory Gregoriadis extended the use of liposomes to the development of liposomal DNA vaccines [].

I myself was lucky enough to have been given the chance of getting a tiny bit involved in Gregorys work about liposomal adjuvants when I was spending 3 months back in 1988 in his laboratory at the Royal Free Hospital in London. Coming from Juergen Laschs laboratory in Halle, Germany I brought with me radiolabeled N -glutaryl-phosphatidylethanolamine (NGPE), which I simply carried in my hand luggage through airport security, an unthinkable security violation under todays travel restrictions. Together with Sascha Klibanov whom I mentioned earlier in connection with PEGylated liposomes, I developed and synthesized NGPE in 1984 in Vladimir Torchilins laboratory in Moscow. We described NGPE as a new hydrophobic anchor for the attachment of proteins to liposomal membranes in FEBS Letters in 1986 [].

Back to Gregorys Nature paper from 1974 [].

As mentioned earlier, Klibanovs work [].

When reflecting on Liposome Technology during the 1990s, another important area of liposome applications for biomedical research with future therapeutic potential has to be highlighted. During the 1980s, numerous attempts have been made to use liposomes for nucleic acid transfer into mammalian cells. Interestingly though, the huge majority of papers describing the use of liposomes for cell transfection were published mainly by one laboratory []. Felgner and his group developed a DNA-transfection protocol that makes use of a synthetic cationic lipid, N -[1-(2,3-dioleyloxy)propyl]- N , N , N -trimethylammonium chloride (DOTMA). The authors found that small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipidDNA complexes with 100 % entrapment of the DNA. They found that DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells which eventually results in both uptake and expression of the DNA. Felgners technique was simple and highly reproducible, and effective for both transient and stable expression of transfected DNA. They reported at that time that depending upon the cell line; lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique. The invention of cationic liposome-forming lipids (which are non-existent in nature) triggered a boom in the area of gene therapy during the 1990s. Cationic liposomes emerged as valuable alternative to viral transfection vectors, gene therapy companies have been founded, and numerous clinical trials have been conducted. However, covering this development which was literally started with Felgners paper in 1987 is beyond the scope of this chapter which I wanted to dedicate to the early days of Liposome Technology, and in particular to the two giants in this field, Alec Bangham and Gregory Gregoriadis.