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Estimation of surface state of poly(ethylene glycol)-coated liposomes using an aqueous two-phase partitioning technique.

Poly(ethylene glycol)-coated liposomes (PEG-liposomes) were prepared from distearoylphosphatidylcholine (DSPC)/cholesterol (Ch) (1:1, molar ratio) with various amounts of distearoyl-N-(monomethoxy poly(ethylene glycol)succinyl)phosphatidylethanolamine (DSPE-PEG). Surface potentials of PEG-liposomes showed negative values, however, the zeta potentials were almost neutral under physiological conditions (150 mM NaCl). Taking these electrical surface properties into consideration, a non-charge-sensitive phase system consisting of 5% PEG8000 and 5% dextran T-500, 0.01 M sodium phosphate, 0.15 M sodium chloride (pH 7.0) was used to estimate the alteration of surface state of PEG-liposomes after interaction with plasma in vitro and in vivo. PEG-liposomes showed increased partitioning to the PEG phase with increasing amount of DSPE-PEG. One hundred percent partitioning to the PEG phase was obtained when 2 or 1 mol% of DSPE-PEG1K or 2K was incorporated into the liposomes, respectively. This PEG/lipid ratio (mol/mol) thus afforded complete protection of the liposomal surface by the PEG moiety. When these PEG-liposomes were incubated with plasma protein (in vitro) or were recovered from liposome-injected mice (in vivo), they showed decreased partitioning to the PEG phase. However, when the in vivo-treated PEG-liposomes were purified by column chromatography and ultracentrifugation, their partitioning to the PEG phase was restored to that of PEG-liposomes incubated in phosphate-buffered saline. Thus although PEG acts as a steric barrier against the attachment of plasma protein to the liposome surface and slows down liposome clearance from the circulation in vivo, a weak interaction remains between PEG-liposome and plasma protein when the incorporated amount of DSPE-PEG is low.