Commentary. Lysosomotropic agents.

Sterically stabilized liposomes

Although the concept of using pharmacologically active macromolecular compounds as drugs is still regarded with much skepticism for both theoretical and practical reasons, interest in this field has grown in recent years because of the opportunity to take advantage of the specific properties of polymeric materials. For low molecular weight drugs, changes in structure often lead to a loss of specific activity. On the other hand, the properties of macromolecular drugs depend on the structure of the polymer used and this can be varied over a wide range by the incorporation of comonomer units, by the application of polymer-analogous reactions, or by related structural changes. A new model is presented for pharmacologically active polymers which incorporates the possibility for continuous variation in (a) solubility and toxicity, (b) fixation and removal of active material, and (c) body distribution. Using the new model as a guide, examples of the synthesis and study of the biological activity of various macromolecular drugs are presented in order to emphasize the importance of this cooperative effort between polymer research and therapeutic problems.

Structure and properties of pharmacologically active polymers

When small, unilamellar lipid vesicles containing a high concentration of the fluorescent dye 6-carboxyfluorescein are incubated with either frog retinas or human lymphocytes, fluroescence distributes widely throughout each cell. Since "self-quenching" largely prevents the dye from fluorescing as long as it remains sequestered in vesicles, it is clear that a considerable amount of dye is released from the vesicles and diluted into the much larger volume of the cell.

http://science.sciencemag.org/content/sci/195/4277/489.full.pdf

Liposome-Cell Interaction: Transfer and Intracellular Release of a Trapped Fluorescent Marker

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4°C Liposomal stability, ie the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum Whole blood and to some extent plasma were less detrimental to stability than was serum (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4°C was maintained for several days, and by 53 days it had declined only moderately Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro

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Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro.

Yeast β-fructofuranosidase (invertase) or 131I-labelled albumin were entrapped into liposomes composed of phosphatidylcholine, cholesterol and phosphatidic acid. Of the β-fructofuranosidase activity in the liposomal preparations 96–100% was latent. The following observations were made in experiments with rats injected with protein-containing liposomes. 1. After injection of β-fructofuranosidase-containing liposomes (220 units or 1.5mg of β-fructofuranosidase and 17.5mg of lipid), β-fructofuranosidase activity in blood retained its latency but the activity declined to 50% of the injected dose in 1h. Within 6h much of this activity was recovered in the liver and spleen (respectively 45% and 10% of that injected). For up to 21h after injection, the mitochondrial–lysosomal fraction was the principal location of the hepatic β-fructofuranosidase activity. 2. Lysosomal localization of liposomal protein was supported by the observed increase in the trichloroacetic acid-soluble radioactivity during incubation of the lysosome-rich fraction of the liver of rats injected with liposomes containing 131I-labelled albumin. 3. Association of liposomal protein with lysosomes was demonstrated on subfractionation of the mitochondrial–lysosomal fraction of the liver of rats injected with β-fructofuranosidase-containing liposomes in a Ficoll–mannitol gradient. β-Fructofuranosidase, lysosomal and mitochondrial enzyme marker activities were found to exhibit similar distribution patterns along the gradient. However, in similar experiments with rats previously injected with Triton WR-1339 or dextran (known to alter the specific gravity of lysosomes), only β-fructofuranosidase and lysosomal marker moved along the gradient, in strikingly similar patterns. 4. The lysosomal localization of injected liposome-entrapped material can probably be utilized in the treatment of certain disorders in man.

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Lysosomal localization of β-fructofuranosidase-containing liposomes injected into rats. Some implications in the treatment of genetic disorders

Liposomes, for many years a tool of the membranologists, have over the last 12 years been used in many other areas of research. Apart from their continuing use in membrane research, including immunological investigations, these phospholipid vesicles have found themselves the centre of attraction as possible carriers of materials of therapeutic interest.

Brenda E. Ryman

Papers

Lysosomal localization of β-fructofuranosidase-containing liposomes injected into rats. Some implications in the treatment of genetic disorders

Liposomes — Further Considerations of Their Possible Role as Carriers of Therapeutic Agents