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Q F Ahkong

Publications and source records attributed to Q F Ahkong.

At least 19 recordsLinked to original sources
PubMed2002

Characterization of the inhibitory effect of PEG-lipid conjugates on the intracellular delivery of plasmid and antisense DNA mediated by cationic lipid liposomes.

Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.

Cell Nucleus↗
PubMed2000

Membrane perturbation and the mechanism of lipid-mediated transfer of DNA into cells.

Mixtures of cationic lipids and unsaturated phosphatidylethanolamine are used extensively for the intracellular delivery of plasmids and antisense oligodeoxynucleotides (ODN) in vitro. However, the mechanism by which cytoplasmic delivery of these large molecules is achieved remains unclear. The common hypothesis is that phosphatidylethanolamine promotes fusion of lipid/DNA particles with endosomal membranes, but this is inconsistent with several reports that have failed to correlate the fusogenic activity of a wide variety of lipid/DNA particles, measured by lipid mixing techniques, with their transfection activity. To address this issue further we have conducted a detailed analysis of the lipid mixing and DNA transfer activity of two, physically similar but functionally different, lipid/DNA particles composed of equimolar dioleyldimethylammonium chloride (DODAC) and dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC). In combination with DODAC both phospholipids form almost identical lipid/DNA particles, they are endocytosed by cells to the same extent and each undergoes equivalent lipid mixing with cell membranes after uptake. Despite this, DNA transfer is 10- to 100-fold more extensive for lipid/DNA particles containing DOPE. We conclude that lipid mixing between lipid-based delivery systems and endosomal membranes must occur for DNA transfer to occur. However, the potency of different lipid/DNA particles correlates better with the ability of the exogenous lipid to disrupt membrane integrity.

Animals↗
PubMed1994

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.

Animals↗
PubMed1993

Divalent cations, phospholipid asymmetry and osmotic swelling in electrically-induced lysis, cell fusion and giant cell formation with human erythrocytes.

We have previously reported that acidic phospholipids are exposed at the surface of human erythrocytes when the cells are subjected to electrical breakdown. It has now been shown that the prothrombinase assay, which was used previously for the determination of acidic phospholipids, is specific for phosphatidylserine under the conditions of our experiments. In the light of this finding, we have investigated and characterised factors that govern cell lysis, cell fusion, and the formation of giant cells induced by electrical breakdown with human erythrocytes in media of low ionic strength. Divalent cations (1.1 mM) protected the cells against haemolysis, in the order Mn2+ > Ca2+ > Ba2+ > Mg2+ >> Zn2+, whereas about 99% of the cells lysed immediately on breakdown in the presence of Na+ or K+ (2.1 mM), or Al3+ (0.95 mM). The lengths of pearl chains of fused erythrocytes formed was similarly greatest with Mn2+ and decreased progressively with Ba2+, Zn2+, Ca2+ and Mg2+. No cell fusion occurred with Na+, K+, or Al3+. It is suggested that interactions with phosphatidylserine, which is exposed at the cell surface by electrical breakdown, may enable Mn2+, Ba2+ and Ca2+ ions to inhibit cell lysis (via membrane resealing) and facilitate cell fusion. Following electrically-induced cell fusion, erythrocytes round-up into giant cells. It has previously been proposed that Ca2+ ions accelerate the rounding-up process. However, data are presented which show that, as with erythrocytes treated with Sendai virus, the formation of rounded, giant cells following cell fusion depends on the osmotic swelling properties of permeabilised erythrocytes. Osmotic swelling may also have induced any hemi-fused cells present to fuse completely. Zn2+ ions anomalously enabled erythrocytes to round-up very rapidly into giant cells following electrical breakdown. This phenomenon may result from an interaction of Zn2+ ions with cysteine groups in membrane proteins, which decreases the immediate loss of ions that occurs when erythrocytes are subjected to electrical breakdown in low-ionic-strength media.

Cadmium↗
PubMed1993

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.

Animals↗
PubMed1992

Nuclear magnetic relaxation dispersion and 31P-NMR studies of the effect of covalent modification of membrane surfaces with poly(ethylene glycol).

Covalent attachment of methoxypoly(ethylene glycol) (MPEG) 5000 to the surface of unilamellar liposomes composed of egg phosphatidylcholine and dioleoylphosphatidylethanolamine (DOPE) (8:2) containing paramagnetic chelates, either entrapped within the interior volume of the liposomes, or associated with the membrane surface, had no effect upon the measured spin-lattice relaxation rates (1/T1) for water in these systems. 31P-NMR studies indicate no destabilization of dioleoylphosphatidylcholine (DOPC)/(DOPE) (1:1) vesicles following attachment of MPEG. However, in DOPC/DOPE (1:3) mixtures, covalent modification with MPEG results in a destabilization of multilamellar vesicles into smaller vesicular structures. These results indicate that covalent attachment of poly(ethylene glycol) to liposomal magnetic resonance agents may prove a useful method for increasing their utility as vascular MR agents by extending their lifetime in the circulation, without decreasing the relaxivity of paramagnetic species associated with the liposome, but that the presence of PEG covalently attached to the membrane surface may modify the polymorphic phase behavior of the lipid system to which it is covalently linked.

Lipid Bilayers↗
PubMed1992

Permeability of liposomal membranes to water: results from the magnetic field dependence of T1 of solvent protons in suspensions of vesicles with entrapped paramagnetic ions.

The diffusive permeability to water molecules, Pd, of lipid vesicles with entrapped paramagnetic solute ions can be determined rapidly from analysis of the magnetic field dependence (nuclear magnetic relaxation dispersion, or NMRD profile) of T1 of exterior solvent water protons. Such data yield tau, the mean lifetime of solvent molecules inside the vesicles, from tau = (fT1Para) - T1Ves, where f is the volume fraction of entrapped water, T1Para is the observed T1 corrected for buffer background, and T1Ves is the relaxation time of water protons in the entrapped solution. For small spherical unilamellar vesicles of inner radius R, Pd = R/3 tau, f can be obtained accurately from knowledge of both the concentration of Gd(DTPA)2- in the solution in which the vesicles were formed and the average concentration of ions in the final sample. At low temperatures, in the limit of slow exchange, T1Para becomes independent of field and tau = fT1Para; the observation of a field-independent profile is a control that confirms that no paramagnetic material is external to the vesicles. We have measured T1Para, using a field-cycling relaxometer, for suspensions of POPC (1-palmitoyl-2-oleoyl-lecithin) vesicles with 100-500 mM entrapped Gd(DTPA)2- and membrane concentrations of cholesterol ranging from 0 to 40 mol %. These profiles, which span the field range 0.01-50 MHz proton Larmor frequency, were taken at 5, 15, 25, and 35 degrees C. Concentrations of Gd(DTPA)2- were determined independently by both ICP analyses and NMRD methods. Values for Pd for vesicles with 100 mM Gd(DTPA)2- and outer diameters 100 nm +/- 20%, as determined by quasielastic light scattering, are 63, 47, 24, 16, and 8.7 x 10(-4) cm s-1, at 25 degrees C, for cholesterol concentrations of 0, 10, 20, 30, and 40%, respectively. The corresponding activation enthalpies are 14, 14, 14, 17, and 17 kcal/M. Comparison with 2H NMR studies of deuterated POPC vesicles with no cholesterol at 20 degrees C, and with 10% at 40 degrees C, which yielded the same order parameter for the palmitoyl acyl chains, gives no indication of a correlation between order parameter and permeability.

Contrast Media↗
PubMed1992

The design of liposomal paramagnetic MR agents: effect of vesicle size upon the relaxivity of surface-incorporated lipophilic chelates.

The 1/T1 NMRD profiles of lipid vesicles with the paramagnetic ion Gd attached via a chelate covalently linked to the membrane surface show a peak at approximately 20 MHz indicating that fluctuations of approximately 10(-8) s contribute to the form of the dispersion profile. If the correlation time for fluctuations of the paramagnetic chelate on the membrane surface is much less than the correlation time for rotation of the lipid vesicle, it would be expected that the measured 1/T1 relaxation rate for solvent protons should be invariant with vesicle size above a certain minimum vesicle diameter. We show that this is indeed the case for vesicles in the size range 50 to 400 nm average diameter and discuss general design considerations for the preparation of vesicle-associated MR contrast agents based upon paramagnetic chelates either trapped within the vesicle interior or attached to the membrane surface.

Chelating Agents↗
PubMed1992

Attachment of 99mTc to lipid vesicles containing the lipophilic chelate dipalmitoylphosphatidylethanolamine-DTTA.

The binding of 99mTc to negatively-charged and neutral unilamellar lipid vesicles was investigated in the absence and presence of the ligand diethylenetriaminepentaacetic acid (DTPA) covalently attached to the headgroup of phosphatidylethanolamine at the surface of the membrane. Even in the absence of DTPA on the membrane surface, 99mTc reduced by Sn bound to the membrane surface but rapidly dissociated from the vesicles in the presence of plasma in vitro. When DTPA was present on the membrane surface, dissociation of 99mTc from the vesicle surface in plasma was much reduced. The dissociation of 99mTc from the surface of negatively-charged vesicles was less than for neutral vesicles in the absence of ligand but was markedly greater than for vesicles containing the ligand DTPA, suggesting that the binding of 99mTc to vesicles with surface-attached DTPA could not be explained solely on the basis of the negative charge provided by the DTPA. In vitro experiments using 14C-labeled lipids as well as in vivo imaging studies indicated that dissociation of 99mTc from the surface of the vesicle did not arise predominantly because of lipid exchange with plasma components or due to cleavage of Tc-DTPA from the vesicle surface. For vesicles with surface-attached DTPA, 99mTc dissociation from the vesicle surface in plasma was further reduced by addition of the antioxidant ascorbate.

Animals↗
PubMed1991

Membrane fusion without cytoplasmic fusion (hemi-fusion) in erythrocytes that are subjected to electrical breakdown.

There are many reports of hemi-fusion in phospholipid vesicles but few published studies on hemi-fusion in cells. We report evidence from both fluorescence microscopy and freeze-fracture electron microscopy for hemi-fusion in the electrofusion of human erythrocytes. We have also characterised the conditions that favour hemi-fusion as opposed to complete fusion, and discuss the possibility that hemi-fusion might precede complete electrically-induced cell fusion. A membrane probe (DiIC16) and a cytoplasmic probe (6-carboxyfluorescein) were used to investigate the behaviour of doubly-labelled human erythrocytes which were aligned in chains by dielectrophoresis and then exposed to high voltage breakdown pulses. Some of the cells were fused by the pulses, as shown by diffusion of both membrane and cytoplasmic probes from labelled to unlabelled cells. With other cells, the membrane probe diffused into unlabelled cells after the breakdown pulses, without the cytoplasmic probe diffusing into unlabelled cells or leaking into the medium. Membrane fusion (hemi-fusion) thus occurred without cytoplasmic fusion in these erythrocytes. Such cells were irreversibly, but fragilely, attached to their neighbours by the breakdown pulses. There was an inverse relationship between conditions that permit complete fusion and those that favour hemi-fusion, with respect to breakdown pulse length, breakdown voltage and, in particular, osmolarity and temperature. The incidence of hemi-fusion in 250 mM erythritol was twice that in 150 mM erythritol, and hemi-fusion was 5-fold greater at 25 degrees C than at 20 degrees C. Hemi-fused erythrocytes occasionally fused completely on heating to 50 degrees C, demonstrating that hemi-fusion can proceed to complete cell fusion. Freeze fracture electron micrographs of preparations of hemi-fused cells revealed long-lived, complementary depressions and protrusions on the E- and P-fracture faces, respectively, of tightly apposed cells that may mediate hemi-fusion. The possibility that the fusion of closely adjacent human erythrocytes by electrical breakdown pulses may involve an intermediate, shared bilayer structure, which is stable in certain conditions but which can be ruptured by osmotic swelling of the permeabilised cells, is discussed.

Cytoplasm↗
PubMed1991

An improved method for the preparation of liposomal gadolinium-DTPA. Ionophore-mediated active entrapment of gadolinium.

We have developed an improved method for the production of liposomal gadolinium-DTPA (Gd-DTPA). The ionophore A23187 facilitates the uptake of externally added Gd into the interior aqueous space of a unilamellar lipid vesicle, where it is chelated by passively entrapped DTPA to form the Gd-DTPA chelate in situ. The presence of a pH gradient across the vesicle membrane is not essential for Gd uptake, the extent of which apparently is limited only by the interior concentration of the chelator. Once formed internally, the Gd-DTPA complex is retained within the vesicles for at least several days at room temperature. Biodistribution studies in mice indicate that liposomal Gd-DTPA prepared by this ionophoretic loading procedure exhibits biodistribution and clearance characteristics similar to 153Gd-DTPA-labeled liposomes prepared by means of passive entrapment of the preformed chelate.

Calcimycin↗
PubMed1991

Detailed toxicity studies of liposomal gadolinium-DTPA.

Acute, subacute, and delayed toxicity testing was assessed in mice for liposomal gadolinium-DTPA (Gd-DTPA), blank liposomes, and nonliposomal Gd-DTPA. In the subacute experiments mice were injected intravenously (IV) with 0.3 mmol/kg Gd-DTPA per day for 30 days in the form of either free Gd-DTPA, liposomal Gd-DTPA, or an equivalent amount of lipid in blank liposomes without Gd-DTPA. The interpolated acute LD50 of liposomal and nonliposomal Gd-DTPA, estimated as a means of identifying the approximate level, was similar (LD50 = 5.7 mmol/kg Gd-DTPA). In subacute toxicity testing, prolonged high doses of liposomal Gd-DTPA caused splenomegaly, cardiomegaly, lymphocytopenia and hypergammaglobulinemia (P less than .05). Nonliposomal Gd-DTPA caused mild cardiomegaly and altered liver enzymes (P less than .05). Blank liposomes caused relatively mild splenomegaly (P less than .05) but few other changes. Delayed testing three months after the subacute testing showed that most of the changes caused by the liposomal Gd-DTPA were reversible.

Animals↗
PubMed1988

Localized osmotic swelling and cell fusion in erythrocytes: possible implications for exocytosis.

Factors that govern the experimentally induced fusion of erythrocytes with one another may generally be relevant to whether or not osmotic forces drive membrane fusion in exocytosis because, under appropriate conditions, osmotic swelling can drive the fusion of erythrocytes. It is now reported that these cells fuse when they are subjected to osmotic swelling caused by exposure to small permeant molecules. The behaviour of erythrocytes in fusion induced by treatment with a concentrated solution of high molecular weight poly(ethylene glycol) (PEG) is also of specific interest in relation to exocytosis because the haemoglobin of erythrocytes that are dehydrated by concentrated solutions of the polymer may be regarded as a model for the tightly packed, dehydrated contents of the granules in secretory cells. We have observed that, under certain conditions of rehydration, the swelling of aqueous microdroplets between the dehydrated haemoglobin and the plasma membrane is closely associated with the fusion of partially rehydrated but still shrunken, PEG-treated erythrocytes. It is therefore apparent that osmotic forces, acting locally at the sites of aqueous microdroplets, can drive the fusion of membranes that encapsulate a dehydrated, concentrated protein, even though gross osmotic swelling at the level of the light microscope is absent. This finding is consistent with the possibility that osmotic swelling may play a role in exocytotic membrane fusion if it is restricted to a small zone immediately under the granule membrane.

Cell Fusion↗
PubMed1987

Movements of fluorescent probes in the mechanism of cell fusion induced by poly(ethylene glycol).

It has been claimed that purified poly(ethylene glycol) (PEG) is able only to aggregate cells and not to fuse them. In our hands, purified PEG 6000 (recrystallized/dialysed) induces both aggregation and fusion of human erythrocytes, and the mechanism of fusion by the purified polymer has been investigated with fluorescent probes. No movement of a carbocyanine probe or of octadecyl rhodamine B chloride from labelled to unlabelled cells occurred in the absence of PEG or with cells treated with concanavalin A, protamine or spermine. With 40% PEG, however, both probes immediately started to diffuse into the membranes of unlabelled cells. This indicates that continuity between the phospholipid bilayer membranes of adjacent erythrocytes (i.e. membrane fusion) is established within seconds in concentrated solutions of the polymer, and precedes the cell fusion event that is induced by purified PEG. These observations are consistent with the idea that micro-regions of shared phospholipid bilayer may be formed in the membranes of cells when they are forced together as a consequence of the dehydrating action of PEG. Intact erythrocytes were cytoplasmically labelled with 6-carboxyfluorescein to avoid the possibility that loading the cells with a cytoplasmic marker by hypotonic haemolysis might modify their response to PEG. Unlike the lipid probes, carboxyfluorescein did not diffuse from labelled to unlabelled cells in the presence of 40% PEG, and there was little diffusion on subsequent dilution of the polymer solution to 13%. However, after the PEG solution had been replaced by an isotonic buffer, a rapid transfer of the cytoplasmic fluorophore to unlabelled cells often occurred. This is considered to be more consistent with the osmotic rupture of a membranous barrier, such as a shared bilayer, between the labelled and unlabelled cells than with the return of cytoplasmic viscosity to normal when the PEG is removed. Possible reasons are discussed for the reported inability of purified PEG to fuse fibroblasts with hypotonically loaded human erythrocytes.

Cell Fusion↗
PubMed1986

Osmotic forces in artificially induced cell fusion.

The importance of cell swelling in the fusion of erythrocytes by three different chemical treatments has been investigated with cells that were cytoplasmically labelled with 6-carboxyfluorescein. Hen erythrocytes, which had been pre-incubated with ionophore A23187 and 5 mM Ca2+ to cause a proteolytic breakdown of the membrane skeleton, were induced to fuse by applying an osmotic shock. Human erythrocytes that had been incubated in an isotonic salt/buffer solution, which was progressively diluted and which contained 0.5 mM La3+ to minimise cell lysis, were also fused. In addition, the fusion of human erythrocytes by 40% poly(ethylene glycol) began only when the poly(ethylene glycol) was diluted, and it mostly occurred when the diluted polymer solution was subsequently replaced by isotonic buffer. In related experiments, the effect of an osmotic gradient on electrically induced cell fusion has been studied. Human erythrocytes in 150 mM erythritol fused more readily than less swollen cells in 200-400 mM erythritol when subjected to a 20 microseconds pulse of 3.5 kV X cm-1, indicating that the extent of cell fusion induced by the breakdown pulse is governed by the combined electrical-compressive and osmotic forces. Since osmotic phenomena are already known to be important in exocytosis, we suggest that these observations on cell fusion indicate that osmotic forces may provide the driving force for many membrane fusion reactions in biological systems.

Animals↗
PubMed1986

An osmotic model for the fusion of biological membranes.

A molecular model for fusion-fission reactions in membranes is proposed that is based on data from studies on artificially induced cell fusion and on the behaviour of phospholipid bilayers: it is put forward as a framework for further investigations into this fundamental property of biological systems.

Animals↗