Kinetic and equilibrium studies of bile salt-liposome interactions.

Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC < C ≈ TC < DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control < MKC < TC ∼ C < DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC < C < DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.

Mechanistic studies of the effect of bile salts on rhodamine 123 uptake into RBE4 cells.

To examine the ability of bile salts (BS) to act as permeation enhancers at the blood brain barrier, the effect of four BS (cholate, deoxycholate, monoketocholate and taurocholate) on accumulation of rhodamine 123 (R123) in rat brain endothelial (RBE4) cells was investigated. Experiments were performed using BS concentrations shown to be noncytotoxic to RBE4 cells. Uptake and efflux of R123 in the absence and presence of BS were studied by fluorescence spectroscopy and confocal microscopy. Changes in RBE4 cell membrane fluidity in the presence of BS were evaluated using fluorescence anisotropy. The direct interaction between BS and R123 (ion pairing) and the effect of BS on distribution of R123 into liposomes were studied by capillary electrophoresis. All BS influenced R123 uptake in a concentration-dependent manner and increased cell membrane fluidity. Monoketocholate produced the greatest increase in uptake and also significantly reduced R123 efflux probably by inhibition of P-glycoprotein (P-gp). Direct interaction of BS and R123 was weak, but distribution of R123 into liposomes was increased by BS. The results suggest that BS increase R123 uptake by increasing cell membrane fluidity and, in the case of MKC, by inhibiting P-gp.

Uptake and release profiles of PEGylated liposomal doxorubicin nanoparticles: A comprehensive picture based on separate determination of encapsulated and total drug concentrations in tissues of tumor-bearing mice.

The PEGylated liposomal nanoparticle has been widely used as a carrier in drug delivery system. To become biologically active, the encapsulated drug must be released from the nanoparticle vehicle. However, due to limitations of current bioanalytical methods, the characterization of this release process has been restricted to determination of total drug in tissues and tumor. As a result, the fate of liposomal nanoparticles including their uptake into target tissue has not been fully characterized. In this study, we developed a novel two-step solid phase extraction on two separated columns procedure to separate liposomes from tissues and tumors without liposomal leakage. This allowed us to determine encapsulated drug, total drug and, by difference, released drug and compare the release and uptake profiles of PEGylated liposomal doxorubicin in tissues and tumor of tumor-bearing mice with corresponding profiles for free doxorubicin. The liposomal nanoparticles released doxorubicin into tumor efficiently and, compared with administration of free drug, increased doxorubicin uptake into tumor by 1.8-fold. It also decreased doxorubicin uptake into heart (0.78-fold lower) with the potential to reduce doxorubicin cardiotoxicity. Drug release reached constant levels in tissues and tumor after 12 h with released doxorubicin concentration remaining at 70-80% of total doxorubicin concentration and in tumor at 86% of total drug concentration. The assay also included determination of the main doxorubicin metabolites. Determination of the metabolites showed that liposomal entrapment delays and decreases the metabolism of doxorubicin but does not alter the metabolic pathway. These results provide a clear and comprehensive picture of the biodistribution of doxorubicin administered in liposomal nanoparticles which may assist in the rational design of other liposomal nanoparticles.