Physicochemical characterization of a PEGylated liposomal drug formulation using capillary electrophoresis.

In this work, the applicability of using CE to perform a physicochemical characterization of a PEGylated liposomal drug formulation of the anti-cancer agent oxaliplatin was examined. Characterization of the liposomal drug formulation using CE instrumentation encompassed: determination of the electrophoretic mobilities, size determination by Taylor dispersion analysis and interaction studies. Electrophoretic mobilities determined by CE were compared with the results obtained by laser Doppler electrophoresis, which were found to be subject to larger variation. Average hydrodynamic diameters of the liposome preparations, as determined by Taylor dispersion analysis, were in the range of 61-84 nm and were compared with the results obtained by dynamic light scattering. Interactions between oxaliplatin (and paracetamol) and the PEGylated liposome were non-detectable by CE frontal analysis as well as by liposome electrokinetic chromatography. In contrast, for the more lipophilic compound propranolol, apparent liposome-aqueous phase distribution coefficients (D(lip) ) were successfully determined by both electrokinetic chromatography (log D(lip) =2.10) and by CE frontal analysis (log D(lip) =2.14). It is envisioned that CE and capillary-based techniques, including Taylor dispersion analysis, will be useful tools for the characterization of nanoparticulate (e.g. liposomal) drug formulations.

Targeting anti-transferrin receptor antibody (OX26) and OX26-conjugated liposomes to brain capillary endothelial cells using in situ perfusion.

Brain capillary endothelial cells (BCECs) express transferrin receptors. The uptake of a potential drug vector (OX26, or anti-transferrin receptor antibody IgG2a) conjugated to polyethyleneglycol-coated liposomes by BCECs was studied using in situ perfusion in 18-day-old rats in which the uptake of OX26 is almost twice as high as in the adult rat. Using radio-labeling, the uptake of OX26 by BCECs after 15-minute perfusion was approximately 16 times higher than that of nonimmune IgG2a (Ni-IgG2a). OX26 and OX26-conjugated liposomes selectively distributed to BCECs, leaving choroid plexus epithelium, neurons, and glia unlabeled. Ni-IgG2a and unconjugated liposomes did not reveal any labeling of BCECs. The labeling of BCECs by OX26 was profoundly higher than that of transferrin. Perfusion with albumin for 15 minutes did not reveal any labeling of neurons or glia, thus confirming the integrity of the blood-brain barrier. The failure to label neurons and glia shows that OX26 and OX26-conjugated liposomes did not pass through BCECs. The expression of transferrin receptors by endothelial cells selective to the brain qualifies OX26 as a candidate for blood-to-endothelium transport. A specifically designed formulation of liposomes may allow for their degradation within BCECs, leading to subsequent transport of liposomal cargo further into the brain.

In vitro and in vivo aspects of N-acyl-phosphatidylethanolamine-containing liposomes.

Incorporation of the phospholipid, N-acyl-phosphatidylethanolamine (NAPE), has shown to increase the liposomal stability towards plasma components in vitro. Besides increasing the circulation-time, NAPE has been shown to contain fusiogenic properties. Hence, fusion between NAPE-liposomes and target cells may be expected, resulting in a favorable delivery of drug to the target cell. In this study, NAPE has been tested as a potential liposomal component of phosphatidylcholine-liposomes. The liposomes were characterized by size, long-term stability and phase transition temperature (T(m)). In vivo behavior of NAPE-liposomes was determined by the blood-circulation half-life in mice. A characterization of the liposomes revealed that high content of NAPE resulted in liposomes of increased size compared to pure phosphatidylcholine-liposomes. However, the liposomes showed only a slight increase in size during storage for 5 weeks. Determination of T(m) for NAPE-liposomes showed increasing values of T(m) by increasing percentage of NAPE in the liposomal bilayer, due to the higher T(m) of NAPE compared to phosphatidylcholine. Blood-clearance studies showed an initial increase in blood-circulation of liposomes containing high amounts of NAPE. Thus, these results suggest that liposomes containing high percentage of NAPE may be a promising candidate for long-circulating liposomes, possibly in combination with other stabilizing components, e.g. cholesterol.

Targeting of liposome-associated calcipotriol to the skin: effect of liposomal membrane fluidity and skin barrier integrity.

Many dermal diseases like psoriasis are characterized by major changes in skin barrier function, which challenge the reproducible delivery of drugs into specific layers of diseased skin. The purpose of this study was to elucidate how liposomal bilayer fluidity and barrier integrity affected the delivery of liposome-associated calcipotriol to the skin. Calcipotriol-containing gel state and liquid state dipalmitoylphosphatidyl-choline:dilauroylphosphatidylcholine liposomes were prepared by extrusion. Using Langmuir monolayers, calcipotriol was shown to affect the packing of the lipid membrane. The penetration of radioactively labeled lipid and calcipotriol into pig skin was examined using the Franz diffusion cell model, and tape stripping was applied to impose an impaired barrier. Distorting the skin barrier resulted in an enhanced penetration of lipid from both gel and liquid state liposomes. In addition, increased penetration of lipid from liquid state liposomes was observed compared to gel state liposomes into barrier-impaired skin. For barrier-impaired skin, an elevated calcipotriol-to-lipid ratio was found in the receptor fluid for both liposome compositions indicating that calcipotriol is released from the vesicles. This suggests that the liposome-mediated delivery of calcipotriol to the epidermis of diseased skin is affected by the fluidity of the liposomal membrane.

Characterization of a liposome-based formulation of oxaliplatin using capillary electrophoresis: encapsulation and leakage.

A capillary electrophoresis-based method to characterize a PEGylated liposomal drug formulation of the anti-cancer agent oxaliplatin was developed. Pharmaceutical characterization in terms of determination of the free and total oxaliplatin concentrations in the liposomal formulation was successfully performed allowing calculation of the percentage of encapsulated drug and encapsulation efficiency. The trapping efficiency was likewise calculated. The capillary electrophoresis method allowed liposome characterization in the intended formulation media (sucrose solution with low electrolyte concentration), and the attained results were consistent with inductively coupled plasma mass spectrometry measurements. Accelerated drug leakage studies were initiated by the sonication of the PEGylated formulation, using an ultrasound probe, subsequently the drug leakage was determined by capillary electrophoresis. The results obtained with the PEGylated liposomes demonstrate that capillary electrophoresis may be a useful tool for the characterization of liposomal drug formulations.

Pig skin structure and transdermal delivery of liposomes: a two photon microscopy study.

In this work we have characterized the architecture and physical properties of pig skin epidermis including its permeability to different liposome formulations. Autofluorescence images show that cells in the epidermis, from the basal layer to the stratum corneum, are organized in clusters that are in turn separated by particular structures we named "canyons". These canyons start in the surface as a wrinkle, eventually closing and going all the way inside the epidermis as a distinct structure that reaches the stratum basale. This structure, described previously in the epidermis of mouse skin as "intercluster pathway", was suggested to be filled with an unknown material and offer low resistance to vesicle penetration. Analysis of LAURDAN Generalized Polarization images of pig skin show that the canyons are filled with a non-polar poorly hydrated material, similar to that observed in pig skin stratum corneum. These results together with the data obtained from skin autofluorescence images suggest that these canyons are invaginations/extension of SC material. Fluorescently labeled lipids incorporated into very flexible liposomes are able to penetrate into the skin, eventually reaching the basal layer and the dermis plane. The presence of charged lipids in the liposomes enhances size stability and thus the efficiency of penetration.