The aminosterol antibiotic squalamine permeabilizes large unilamellar phospholipid vesicles

Abstract

The ability of the shark antimicrobial aminosterol squalamine to induce the leakage of polar fluorescent dyes from large unilamellar phospholipid vesicles (LUVs) has been measured. Micromolar squalamine causes leakage of carboxyfluorescein (CF) from vesicles prepared from the anionic phospholipids phosphatidylglycerol (PG), phosphatidylserine (PS), and cardiolipin. Binding analyses based on the leakage data show that squalamine has its highest affinity to phosphatidylglycerol membranes, followed by phosphatidylserine and cardiolipin membranes. Squalamine will also induce the leakage of CF from phosphatidylcholine (PC) LUVs at low phospholipid concentrations. At high phospholipid concentrations, the leakage of CF from PC LUVs deviates from a simple dose-response relationship, and it appears that some of the squalamine can no longer cause leakage. Fluorescent dye leakage generated by squalamine is graded, suggesting the formation of a discrete membrane pore rather than a generalized disruption of vesicular membranes. By using fluorescently labeled dextrans of different molecular weight, material with molecular weight </=4000 g/mol is released from vesicles by squalamine, but material with molecular weight >/=10,000 is retained. Negative stain electron microscopy of squalamine-treated LUVs shows that squalamine decreases the average vesicular size in a concentration-dependent manner. Squalamine decreases the size of vesicles containing anionic phospholipid at a lower squalamine/lipid molar ratio than pure PC LUVs. In a centrifugation assay, squalamine solubilizes phospholipid, but only at significantly higher squalamine/phospholipid ratios than required for either dye leakage or vesicle size reduction. Squalamine solubilizes PC at lower squalamine/phospholipid ratios than PG. We suggest that squalamine complexes with phospholipid to form a discrete structure within the bilayers of LUVs, resulting in the transient leakage of small encapsulated molecules. At higher squalamine/phospholipid ratios, these structures release from the bilayers and aggregate to form either new vesicles or squalamine/phospholipid mixed micelles.

Modulating membrane binding of α-synuclein as a therapeutic strategy

“…Squalamine exits cells and is cleared from circulation within hours, and dosing and toxicology are established for treating pathological angiogenesis in humans (24). However, squalamine cannot enter the brain after systemic administration, has been shown to result in permeabilization of large phospholipid vesicles (25) and altered liposome fusion, diameter, and melting temperature (5), and may have nonspecific effects on other proteins that associate with membranes by electrostatic forces. Nonetheless, the study by Perni et al (5) reveals a striking effect of squalamine on reducing α-synuclein-mediated aggregation and toxicity in vitro and in vivo.…”

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential

“…Recently, squalamine was discovered to enter cells and cause displacement of proteins that are associated through electrostatic interactions with the inner face of the cytoplasmic membrane (12)(13)(14). Squalamine (3β-N-1-{N-[3-(4-aminobutyl)]-1,3-diaminopropane)-7α, 24R-dihydroxy-5α-cholestane 24 sulfate; molecular weight = 628) (2) carries a net positive charge by virtue of its spermidine moiety and exhibits a high affinity for anionic phospholipids (15,16); on entry into a eukaryotic cell, it neutralizes the negative charge of the surface to which it binds (12,13). Surprisingly, this disruption of electrostatic potential can occur without obvious structural damage to the cell membrane as measured by changes in permeability (13).…”

Identification of squalamine in the plasma membrane of white blood cells in the sea lamprey, Petromyzon marinus

“…Squalamine is believed to disrupt the bacterial membrane by forming a channel and causing the leakage of cellular material [similar to other cationic antimicrobial peptides (35)]. The squalamine-triggered release of fluorescent dyes trapped within phospholipid vesicles observed by Selinsky et al (36) indicates a possible membrane-associated antimicrobial action for this compound. In this study of the sea lamprey, squalamine did not seem to disrupt the white blood cell membranes.…”