Derek Fisher

Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly(ethylene glycol)-coated vesicles.

Well-defined liposome systems have previously established the influence of size, surface charge lipid composition and surface ligands, on in vivo fate and behaviour of model compounds entrapped in liposomes. In the present study, preformed liposomes which quantitatively retain aqueous markers were covalenty coupled via dipalmitoylphosphatidyl-ethanolamine, to the hydrophilic polymer, monomethoxypoly(ethylene glycol) (MPEG 5000). Such liposomes retain the coating in the presence of plasma, and appear to adsorb plasma components more slowly than liposomes without the polymer, shown using an aqueous two-phase partitioning technique. MPEG-coupled liposomes were cleared from the blood circulation up to 30% more slowly than liposomes without MPEG after intravenous administration to mice, despite the unmodified liposomes being of a composition and size shown previously to favour achievement of maximum half-life. It is suggested that the polymer acts as a surface barrier to plasma factors which otherwise bind to liposomes in the blood and accelerate vesicle removal.

99mTc-labeling of lipid vesicles containing the lipophilic chelator PE-DTTA: Effect of tin-to-chelate ratio, chelate content and surface polymer on labeling efficiency and biodistribution behavior

When injected intravenously, lipid vesicles labeled with 99mTc by means of a lipophilic chelator dipalmitoylphosphatidylethanolamine-diethylenetriaminetetraacetic acid (PE-DTTA) are rapidly accumulated by the mononuclear phagocytic system (MPS). By derivatizing the membrane surface with the lipid-polymer complex dipalmitoylphosphatidylethanolamine-monomethoxy polyethylene glycol 5000 (PE-MPEG), MPS uptake can be suppressed and loss of 99mTc label from the lipid surface reduced depending upon both PE-DTTA and PE-MPEG content. For vesicles containing 20% PE-DTTA, addition of PE-MPEG makes no difference to their rate of clearance from the circulation. However for vesicles containing 2% PE-DTTA, addition of more than 0.8% PE-MPEG increases circulation half-life, suppresses liver uptake and reduces renal clearance of the 99mTc label. The molar ratio of reducing agent (Sn) to chelator (PE-DTTA) is critical to efficient and reproducible labeling. For vesicles containing 2% PE-DTTA at a lipid concentration of 100 mM, a Sn/DTTA ratio of 0.35 gives close to optimal labeling. Variation in the Sn/DTTA ratio by a factor of two negatively impacts upon both labeling efficiency in vitro and circulation half-life in vivo. Potential uses for technetium-labeled lipid vesicles with extended circulation half-life are discussed.

Polymer-derivatized technetium 99mTc-labeled liposomal blood pool agents for nuclear medicine applications

By using the lipophilic chelator, dipalmitoylphosphatidylethanolamine-diethylenetriaminetetraacetic acid (DPPE-DTTA), lipid vesicles may be prepared labeled on their surface with technetium 99m. When technetium-labeled vesicles were injected intravenously into rabbits, the half-life for clearance of the label from the circulation was less than 30 min. By further incorporating a synthetic phosphatidylethanolamine-monomethoxypoly(ethylene glycol) 5000 conjugate (PE-MPEG) the circulation half-life of the radiolabel was increased, liver uptake decreased and exchange of technetium from the vesicle surface suppressed, depending upon both the DPPE-DTTA and PE-MPEG content. For vesicles containing 20 mol% DPPE-DTTA, incorporation of PE-MPEG had no effect upon the circulation half-life of the radiolabel, however, for vesicles containing 2 mol% DPPE-DTTA, incorporation of more than 4 mol% PE-MPEG increased the circulation half-life of the label to more than 12 h. Less than 2 mol% PE-MPEG or 8 mol% ganglioside GM1 were, however, ineffective at increasing the circulation half-life of surface-bound technetium. It was shown that unilamellar lipid vesicles with DPPE-DTTA can be lyophilized in the presence of external sucrose, subsequently rehydrated with no change in vesicle size and labeled with technetium. It is suggested that polymer-derivatized, technetium-labeled vesicles may prove a useful substitute for technetium-labeled red blood cells as a vascular marker in various nuclear medicine procedures and that lyophilization/rehydration provides a possible route to realization of such vesicles in a pharmaceutically useful form.

Analysis and purification of monomethoxy-polyethylene glycol by vesicle and gel permeation chromatography

Abstract Vesicle chromatography (VC) and gel permeation chromatography (GPC) were used for characterisation and purification of monomethoxy-polyethylene glycol (M-PEG), a reagent for protein modification. Detection of low concentrations of contaminating PEG was facilitated by a very sensitive colourimetric detection method with a detection limit of 1 μg/ml. For analytical purposes GPC on Superose 12 was superior to VC. Molecular masses, polydispersity and percentage of contaminating PEG were estimated. As a comparison 1H NMR spectroscopy was carried out. The results were in good accordance with GPC. A two-step preparative purification with VC of M-PEG containing 22.9% PEG reduced the PEG content to 4.4%.

Phase partitioning detects differences between phospholipase-released forms of alkaline phosphatase — a GPI-linked protein

A number of enzymes are known to release alkaline phosphatase and other glycan phosphatidylinositol-anchored proteins from membrane surfaces. We describe a novel approach to detect and measure these activities by partitioning in aqueous phase systems. The procedures avoid the complications of micelle-formation involving hydrophobic molecules that may arise with detergent-based partition systems and can clearly distinguish between inositol-specific phospholipase C and D activities.

A method for the purification of DNA/protein complexes applied to DNA topoisomerase II cleavage sites

The object of this study was to devise a purification method for DNA/topoisomerase II complexes, with which to examine the enzymes cleavage site specificity in cellular differentiation. Retinoic acid-induced differentiation involves topoisomerase II-mediated transient changes in DNA supercoiling, but it is not known whether this occurs at specific sites in the genome. Topoisomerase II forms a covalent DNA enzyme complex as it acts, which can be recovered by the sodium dodecyl sulfate (SDS)/KCl precipitation method, but this method fails to recover significantly more DNA from cells induced to differentiate. This may in part reflect the low numbers of retinoic acid-induced protein-linked breaks in DNA and also the methods relative inefficiency for DNA with few attached topoisomerase molecules. This suggested that an additional purification method would be required to enrich sufficiently for cleavage site DNA to address the issue of site specificity. The principle of our method is to couple poly(ethylene glycol) (PEG) to topoisomerase while it is covalently attached to DNA and then to use phase partitioning in an aqueous two-phase system of PEG and phosphate to separate free DNA from DNA bound to PEG-modified topoisomerases (which have high affinities for the phosphate-rich and PEG-rich phases, respectively). The method can be used in conjunction with DNase protection and, unlike the SDS/KCl method, can fractionate short fragments of DNA to which single protein molecules are attached. Using the SDS/KCl precipitation and new method in series, we have recovered protein-linked DNA from HL60 cells induced to differentiate to the granulocyte lineage (by retinoic acid) or to the monocyte/macrophage lineage (by phorbol myristate acetate) and have demonstrated that specific sequences become protein linked, probably to topoisomerase II, during induced differentiation.

Separation of alkaline phosphatase isoforms with and without intact glycan-phosphatidylinositol anchors in aqueous polymer phase systems.

Alkaline phosphatase (ALP) isoforms can be distinguished from each other by their partition characteristics in aqueous two-phase systems composed of water-soluble polymers, the phases of which are differentially sensitive to the presence of glycan-phosphatidylinositol anchors. Compared with detergent-based systems, the aqueous polymer systems have the advantage that micelle formation does not take place. Partition of anchor-intact and anchor-degraded molecules in the latter systems is further improved by attachment of a hydrophobic ligand to one of the phase forming polymers. In this way, anchor-intact molecules can be separated from molecules with degraded anchors in a single partition step. The method has been used to confirm that ALP in human serum is predominantly anchor degraded, whereas in bile it retains its anchor intact.

Interfacial Events in Phase Separation and Cell Partitioning in Aqueous Two-Phase Systems

The processes by which phase separation occurs in aqueous two-phase systems of polyethylene glycol and dextran are described in terms of the fluid dynamics of phase droplets and phase streams. Interactions of cells with the surfaces of these microphases early in phase separations determine the cell partition coefficient, which is conventionally measured when phase separation is virtually completed.

A New Approach to Countercurrent Distribution Combining Separation with Analysis in the Biomek Automated Laboratory Workstation

A method of programming the Biomek Automated Laboratory Workstation has been developed to perform countercurrent distribution separations of both solutes and cells using aqueous two-phase systems. Studies involving an 11-transfer CCD show good correlation between experimental and theoretical distributions. The separation of high and low partition coefficient cells have shown that the Biomek’s mixing and transfer regimes work well for both particulates and solubles. These results could form the basis of a new approach to the separation of viable biological material and extend the range of an already versatile piece of laboratory equipment.