Analytical Sciences

Precise Size Determination of Amphotericin B and Nystatin Channels Formed in Erythrocyte and Liposomal Membranes Based on Osmotic Protection Experiments

Takashi KATSU,* Shiho OKADA,* Tomonori IMAMURA,* Keiko KOMAGOE,* Kazufumi MASUDA,** Tsuyoshi INOUE,* and Satoshi NAKAO*

* Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan
** School of Pharmacy, Shujitsu University, Nishigawara, Okayama 703-8516, Japan

The colloid osmotic nature of the cell lysis can be prevented by adding osmotic protectants of appropriate sizes to the outer medium. We introduced inorganic and organic electrolytes as protectants to determine the precise channel sizes of the polyene antibiotics, amphotericin B and nystatin, in addition to the sugars so far widely used for this purpose. Because colloid osmotic cell lysis is evidenced by the loss of membrane permeability barriers for small sizes of ions, such as K⁺, preceding hemolysis, we firstly simultaneously monitored the time response of the K⁺ efflux and hemolysis induced by amphotericin B by combining a fiber-optic spectrometer with a K⁺-selective electrode. Based on this experiment, we evaluated the sizes of channels of the polyene antibiotics formed in the erythrocyte membrane using the radii of hydrated ions calculated from a modified Stokes' law, as well as the radii of sugars. The radii of channels formed by amphotericin B and nystatin were found to be in a very narrow range of 0.36 - 0.37 nm. Similar experiments were performed using calcein-loaded liposomes containing cholesterol or ergosterol, and the radii of channels formed in these liposomal membranes were also found to be the same as when formed in an erythrocyte membrane. The present results demonstrated that introducing the sizes of hydrated ions can afford a more precise channel size than the use of sugars alone.

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