Jeffery J. Wheeler

Stabilized plasmid-lipid particles: construction and characterization

A detergent dialysis procedure is described which allows encapsulation of plasmid DNA within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating. These ‘stabilized plasmid-lipid particles’ (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid per particle and fully protect the encapsulated plasmid from digestion by serum nucleases and E. coli DNase I. Encapsulation is a sensitive function of cationic lipid content, with maximum entrapment observed at dioleoyldimethylammonium chloride (DODAC) contents of 5 to 10 mol%. The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of ‘fusigenic’ lipids such as dioleoylphosphatidylethanolamine (DOPE). The in vitro transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the PEG polymer to the surface of the SPLP. Shorter acyl chain lengths result in a PEG coating which can dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity. It is suggested that SPLP may have utility as systemic gene delivery systems for gene therapy protocols.

Accumulation of doxorubicin and other lipophilic amines into large unilamellar vesicles in response to transmembrane pH gradients

The uptake of the anticancer agent doxorubicin into large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient (inside acidic) has been investigated using both kinetic and equilibrium approaches. It is shown that doxorubicin uptake into the vesicles proceeds via permeation of the neutral form and that uptake of the drug into LUVs with an acidic interior is associated with high activation energies (Ea) which are markedly sensitive to lipid composition. Doxorubicin uptake into egg-yolk phosphatidylcholine (EPC) LUVs exhibited an activation energy of 28 kcal/mol, whereas for uptake into EPC/cholesterol (55:45, mol/mol) LUVs Ea = 38 kcal/mol. The equilibrium uptake results obtained are analyzed in terms of a model which includes the buffering capacity of the interior medium and the effects of drug partitioning into the interior monolayer. From the equilibrium uptake behaviour, a doxorubicin partition coefficient of 70 can be estimated for EPC/cholesterol bilayers. For a 100 nm diameter LUV, this indicates that more than 95% of encapsulated doxorubicin is partitioned into the inner monolayer, presumably located at the lipid/water interface. This is consistent with 13C-NMR behaviour as a large proportion of the drug appears membrane associated after accumulation as reflected by a broadening beyond detection of the 13C-NMR spectrum. The equilibrium accumulation behaviour of a variety of other lipophilic amines is also examined in terms of the partitioning model.

The cationic lipid stearylamine reduces the permeability of the cationic drugs verapamil and prochlorperazine to lipid bilayers: Implications for drug delivery

The therapeutic activity of a wide variety of drugs is significantly improved when their longevity in the circulation is extended by encapsulation in liposomes. To improve the retention of cationic drugs in liposomes, we have investigated the effect of the cationic lipid stearylamine on the permeability of the calcium channel blocker verapamil and the antipsychotic drug prochlorperazine, both of which are also multidrug resistance modulators. Both drugs were efficiently incorporated into liposomes composed of DSPC/cholesterol that possessed a transmembrane pH gradient (inside acidic). However, the efflux of the loaded drugs was relatively rapid (i.e., 50% of the encapsulated verapamil was released after 4 h at 37 degrees C), despite the presence of a 3 unit pH gradient (pHi = 4.0, pHo = 7.5). Drug retention within the liposomes was improved by increasing the magnitude of the transmembrane pH gradient to approx. 5 units (pHi = 2.0, pHo = 7.5). Further improvements in drug retention were achieved by the addition of 10 mol% of the cationic lipid stearylamine in the DSPC/cholesterol liposomes. The combination of the 5 unit pH gradient and stearylamine resulted in increases of the retention of verapamil and prochlorperazine by approx. 20- and 5-fold, respectively. Calculation of the permeability coefficients for the charged (cationic) and neutral forms of the drugs indicated that the neutral forms of both drugs were approx. 10(4)-fold more permeable than were the cationic forms of the drugs. Further, the presence of stearylamine reduced the permeability coefficient for the cationic species of the drugs by approximately an order of magnitude, but had no effect on the neutral species of the drugs. The efflux curves observed for both verapamil and prochlorperazine could be mathematically modeled by assuming that the primary influence of stearylamine was on the development of a positive surface charge density on the inner monolayer of the liposome. Taken in sum, these results indicate that stearylamine is effective at decreasing the leakage of cationic drugs from liposomes, and may prove to be a valuable component of liposomal drug formulations.

Ionophore-mediated loading of Ca2+into large unilamellar vesicles in response to transmembrane pH gradients

The Ca2+ translocating properties of the carboxylic ionophores A23187, ionomycin and lasolocid A (X537A) have been investigated by employing large unilamellar vesicles that exhibit a pH gradient (acidic interior). An analysis of Ca2+ uptake at equilibrium reveals that Ca2+ accumulation is an electroneutral process, whereby one Ca2+ ion is transported in for every two H+ ions transported out. A kinetic analysis shows that both A23187 and ionomycin transport Ca2+ in the form of a 1:1 cation:ionophore complex, whereas a 1:2 complex is observed for lasolocid A. The specificity of the ionophores for transporting Ca2+ is reflected by the influence of exterior Na+ ions that inhibit Ca2+ uptake for lasolocid A but do not influence ionomycin-mediated uptake.

Identification of carrot inositol phospholipids by fats atom bombardment mass spectrometry

Inositol phospholipids from carrot cell membranes grown in suspension cultured were purified by thinlayer chromatography (TLC) or column chromatography and tentatively identified by co-migration on TLC with animal inositol phospholipid standards. For more rigorous chemical characterization, carrot inositol phospholipids were then analyzed by negative ion fast atom bombardment mass spectrometry (FABMS). One phosphatidylinositol (PI), two lysophosphatidylinositols (LPI), and one phosphatidylinositol monophosphate (PIP) were identified in the carrot samples by the observation of ions [M-H]− and numerous fragment ions in the negative FAB mass spectra. MS/MS analysis were carried out to obtain further structural information of these phospholipids using a double-focusing mass spectrometer in which the magnetic sector (B) and the electrostatic analyzer (E) were scanned at a constant ratio (B/E). These B/E linked scans provided fragment ions of selected precursor ions while eliminating matrix and other contaminating ions. No molecular ions were detected for lysophosphatidylinositol monophosphate (LPIP) or phosphatidylinositol bisphosphate (PIP2), but fragment ions corresponding to these structures were observed. The primary fatty acids present in the carrot inositol phospholipids were linoleic (18∶2) and palmitic (16∶0) acids, whereas animal lipids contained arachidonic (20∶4), stearic (18∶0), linoleic, and palmitic acids. The only phosphatidylinositol found in carrot cells was palmitoyl linoleoyl PI.

Generation and characterization of iron- and barium-loaded liposomes

Previous work (Veiro and Cullis (1990) Biochim. Biophys. Acta 1025, 109-115) has shown that Ca2+ can be accumulated into large unilamellar vesicles (LUVs) in the presence of a transmembrane pH gradient (inside acidic) and the Ca(2+)-ionophore A23187. Here, the ability of A23187 to mediate the uptake of iron and barium into LUVs has been investigated. It is shown that under appropriate conditions of temperature and A23187 concentration, iron (in the form of Fe2+) can be accumulated into EPC and DSPC/cholesterol (55:45; mol/mol) LUVs with an acidic interior. This uptake is dependent on the internal buffer concentration, with maximum levels of uptake in the range of 300 nmol of cation per mumol lipid. The DSPC-cholesterol LUV systems exhibit superior retention properties compared to the EPC systems. It is demonstrated that Ba2+ can also be loaded by similar methods. It is also shown that the maximally loaded Fe(2+)- and Ba(2+)-containing LUVs exhibit increased densities. This is expressed by enhanced gravimetric properties, as an increased proportion of the loaded LUVs can be pelleted by low speed centrifugation, and by enhanced electron densities, in that the Ba(2+)-loaded systems can be directly visualized employing cryo-electron microscopy.

The presence of sn-1-palmitoyl lysophosphatidylinositol monophosphate correlates positively with the fusion-permissive state of the plasma membrane of fusogenic carrot cells grown in suspension culture

Two analogues of lysophosphatidylinositol monophosphate (LPIP) occur in fusogenic carrot cells and protoplasts and are distinguished from each other on thin-layer plates by differing R t values. The lower R f LPIP comigrates with sn -1-palmitoyl LPIP (16:0 LPIP) synthesized from sn -1-palmitoyl lysophosphatidylinositol. The upper R f LPIP is presumed to be sn -2-linoleoyl LPIP (18:2 LPIP) based on fatty acid analysis of phosphatidylinositol monophosphate (PIP) and comigration with the upper R f product resulting from the base-catalyzed hydrolysis of PIP. The 18:2 analogue is only a minor component of the LPIP recovered from the fusogenic cells and protoplasts, but it is the predominant form of LPIP in nonfusogenic cells and protoplasts. LPIP is found primarily in the plasma membranes of these cells representing from 10 to 20% of the total [ 3 H]inositol-labeled lipid recovered from isolated plasma membrane. Even though the fusogenic cells lose some of the LPIP as a result of cell wall digestion, if 2% or more of the total protoplast inositol lipid is LPIP, the protoplasts fuse spontaneously with a fusion frequency of greater than 60%. As the fusogenic protoplasts lose their fusion potential, they also lose the relative amount of 16:0 LPIP. Inhibiting the in vivo metabolism of phosphatidylinositol bisphosphate (PIP 2 ) and PIP in cells and protoplasts with the aminoglycoside, neomycin, does not inhibit fusion. These data indicate that the presence and metabolism of 16:0 LPIP but not the polyphosphoinositides, PIP and PIP 2 , correlate positively with protoplast fusion.