pH gradient loading of anthracyclines into cholesterol-free liposomes: enhancing drug loading rates through use of ethanol

Abstract

Application of cholesterol-free liposomes as carriers for anticancer drugs is hampered, in part, because of standard pH gradient based loading methods that rely on incubation temperatures above the phase transition temperature (Tc) of the bulk phospholipid to promote drug loading. In the absence of cholesterol, liposome permeability is enhanced at these temperatures which, in turn, can result in the collapse of the pH gradient and/or unstable loading. Doxorubicin loading studies, for example, indicate that the drug could not be loaded efficiently into cholesterol-free DSPC liposomes. We demonstrated that this problem could be circumvented by the addition of ethanol as a permeability enhancer. Doxorubicin loading rates in cholesterol-free DSPC liposomes were 6.6-fold higher in the presence of ethanol. In addition, greater than 90% of the added doxorubicin was encapsulated within 2 h at 37 degrees C, an efficiency that was 2.3-fold greater than that observed in the absence of ethanol. Optimal ethanol concentrations ranged from 10% to 15% (v/v) and these concentrations did not significantly affect liposome size, retention of an aqueous trap marker (lactose) or, most importantly, the stability of the imposed pH gradient. Cryo-transmission electron micrographs of liposomes exposed to increasing concentrations of ethanol indicated that at 30% (v/v) perturbations to the lipid bilayer were present as evidenced by the appearance of open liposomes and bilayer sheets. Ethanol-induced increased drug loading was temperature-, lipid composition- and lipid concentration-dependent. Collectively, these results suggest that ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of cholesterol-free liposomes at temperatures below the Tc of the bulk phospholipid.

Enhanced bactericidal potency of nanoliposomes by modification of the fusion activity between liposomes and bacterium

“…It is known that cholesterol-containing vesicles reduce vesicular content leaking and fusion in artificial liposomal systems. [29][30][31][32] The results reported here are the first systematic investigation of the effects of cholesterol on liposome-bacterium fusion. Cholesterol is the predominant sterol in the mammal cell membranes.…”

Determination of intraliposomal pH and its effect on membrane partitioning and passive loading of a hydrophobic camptothecin, DB-67

“…Trans-membrane pH gradients are also used for the purpose of drug loading into vesicles during their formulation (Dos Santos et al, 2004;Madden et al, 1990). The maintenance of established pH gradients both in vitro and in vivo may be critical to ensure efficient drug loading and retention.…”

A unique squalenoylated and nonpegylated doxorubicin nanomedicine with systemic long-circulating properties and anticancer activity

“…Thus, there is a consensus in the drug-delivery community that new concepts are needed for the construction of long-circulating nanocarriers avoiding the use of PEG. Another concern is the low drug-loading (i.e., milligrams of the anticancer anthracycline per milligrams of carrier material), though the use of tansmembrane pH gradient-loading methods into liposomes varies from 0.05 μM/1 μM lipid to 0.21 μM/1 μM lipid (25,26). With polymer nanoparticles, the maximum payload is generally less than 10% (wt/wt) (27).…”