Kunikazu Moribe

Molecular localization and state of amphotericin B in PEG liposomes

We investigated the molecular localization and state of amphotericin B (AmB) encapsulated in polyethylene glycol (PEG)-coated liposomes. AmB-encapsulating PEG-liposomes composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (CH) and distearoyl-N-(monomethoxy poly(ethylene glycol)succinyl) phosphatidylethanolamine (DSPE-PEG, average MW of the PEG chain 2000) were prepared by hydration with 9% sucrose solution and extrusion. The amount of AmB encapsulated in the liposomes increased with incorporation of DSPE-PEG and decreased with that of CH. The molecular localization and state of AmB were investigated by PEG/dextran two-phase partition, potassium permeability measurement, fluorescence quenching measurement and circular dichroism (CD) spectroscopy. The results suggest that there are two types of AmB localization in PEG-liposomes, one of which corresponds to the complex of AmB with DSPE-PEG on the membrane surface, while the other corresponds to the pore form of AmB in the hydrophobic core of the liposomal membrane. AmB in PEG liposomes was present in both aggregated and monomeric states.

Enhanced Encapsulation of Amphotericin B into Liposomes by Complex Formation with Polyethylene Glycol Derivatives

AbstractPurpose. A highly efficient method was developed for the encapsulation of amphotericin B (AmB) in liposomes, and the mechanism involved was characterized. Methods. AmB was encapsulated in dipalmitoylphosphatidylcholine/cholesterol (DPPC/CH, 2:1) liposomes after complex formation with distearoyl-N-(monomethoxy poly(ethylene glycol) succinyl) phosphatidylethanolamine (DSPE-PEG). Hydration of lipids was done with 9% sucrose solution. Results. The encapsulated amount of AmB was 111 μg/mg lipid, which was much higher than that obtained by the same method without DSPE-PEG (14 μg/mg lipid). The amount encapsulated increased with amount of DSPE-PEG used and with PEG molecular weight. Encapsulation efficacy was also influenced by the type of PEG derivatives used and by the modification of AmB, suggesting the involvement of complex formation between AmB and DSPE-PEG. Absorption and 31P-NMR spectral analyses indicated that interactions between the amino and phosphate groups and between the polyene and PEG moieties in AmB and DSPE-PEG, respectively, play an important role in the complex formation. Conclusions. Complex formation of AmB with DSPE-PEG allows the highly efficient encapsulation of the drug in liposomes. This simple technique should be applicable to other hydrophobic drugs.

Spectroscopic investigation of the molecular state of nystatin encapsulated in liposomes.

The stability and spectral properties of nystatin-encapsulating liposomes, composed of various combinations of dipalmitoyl phosphatidylcholine (DPPC), cholesterol (CH) and distearoyl-N-(monomethoxy poly(ethylene glycol)succinyl) phosphatidylethanolamine (DSPE-PEG), were studied in order to elucidate the molecular state and localization of nystatin encapsulated in liposomes. Localization of nystatin at the surface region of the liposomal membrane was investigated by PEG/dextran two-phase partition and measurement of the fluorescence quenching of nystatin by p-xylene-bis-pyridinium bromide (DPX). In DPPC/DSPE-PEG liposomes and DPPC/CH/DSPE-PEG liposomes, containing 151 and 160 mcg nystatin per mg lipid, respectively, nystatin appeared to be present at the surface region of the liposomal membranes. Self-quenching of nystatin fluorescence was observed in DPPC/CH and DPPC/CH/DSPE-PEG liposomes even at low encapsulated amounts, suggesting the localization of nystatin in CH-incorporating membranes. In CH-free liposomes, nystatin molecules were at first delocalized in the membranes and then self-associated at a higher level of encapsulation. Absorption and circular dichroism (CD) spectra were also measured to examine the monomeric and aggregated states of nystatin in liposomes. High encapsulation efficacy was observed in DPPC and DPPC/DSPE-PEG liposomes, but the highest stability and retention of nystatin in liposomes were observed in DPPC/CH/DSPE-PEG liposomes, evaluated in terms of the nystatin and calcein release from nystatin-encapsulating liposomes in vitro. From the results, possible encapsulation mechanisms of nystatin in liposomes narrowed down to the following three points; interaction with lipid membrane, adsorption on the liposomal surface and complex formation with DSPE-PEG.