Stabilized plasmid-lipid particles: pharmacokinetics and plasmid delivery to distal tumors following intravenous injection.

A previous study has shown that plasmid DNA can be encapsulated in lipid particles (SPLP, "stabilized plasmid lipid particles") of approximately 70 nm diameter composed of 1,2-dioleoyl-3-phosphatidyl-ethanolamine (DOPE), the cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and poly(ethylene glycol) conjugated to ceramide (PEG-Cer) using a detergent dialysis process (Wheeler et al. (1999) Gene Therapy 6, 271-281). In this work we evaluated the potential of these SPLPs as systemic gene therapy vectors, determining their pharmacokinetics and the biodistribution of the plasmid and lipid components. It is shown that the blood clearance and the biodistribution of the SPLPs can be modulated by varying the acyl chain length of the ceramide group used as lipid anchor for the PEG polymer. Circulation lifetimes observed for SPLPs with PEG-CerC14 and PEG-CerC20 were t(1/2) = approximately 1 and approximately 10 h, respectively. The SPLPs are stable while circulating in the blood and the encapsulated DNA is fully protected from degradation by serum nucleases. The accelerated clearance of SPLPs with PEG-CerC14 is accompanied by increased accumulation in liver and spleen as compared to PEG-CerC20 SPLPs. Delivery of intact plasmid to liver and spleen was detected. Significant accumulation (approximately 10% of injected dose) of the long circulating SPLPs with PEG-CerC20 in a distal tumor (Lewis lung tumor in the mouse flank) was observed following i.v. application and delivery of intact plasmid to tumor tissue at approximately 6% injected dose/g tissue is demonstrated.

Lipid-based systems for the intracellular delivery of genetic drugs.

Currently available delivery systems for genetic drugs have limited utility for systemic applications. Cationic liposome/plasmid DNA or oligonucleotide complexes are rapidly cleared from circulation, and the highest levels of activity are observed in 'first pass' organs, such as the lungs, spleen and liver. Engineered viruses can generate an immune response, which compromises transfection resulting from subsequent injections and lack target specificity. A carrier, which can accumulate at sites of diseases such as infections, inflammations and tumours, has to be a small, neutral and highly serum-stable particle, which is not readily recognized by the fixed and free macrophages of the reticuloendothelial system (RES). This review summarizes lipid-based technologies for the delivery of nucleic acid-based drugs and introduces a new class of carrier systems, which solve, at least in part, the conflicting demands of circulation longevity and intracellular delivery. Plasmid DNA and oligonucleotides are entrapped into lipid particles that contain small amounts of a positively charged lipid and are stabilized by the presence of a polythylene glycol (PEG) coating. These carriers protect nucleic acid-based drugs from degradation by nucleases, are on average 70 nm in diameter, achieve long circulation lifetimes and are capable of transfecting cells.

pH-induced destabilization of lipid bilayers by a lipopeptide derived from influenza hemagglutinin.

A synthetic twenty-one amino acid peptide (AcE4K) based on the amino acid sequence of the influenza HA2 fusion peptide was coupled to a distearoylglycerol lipid anchor by amidation of an N-terminal lysine side chain. The secondary structure of Lipo-AcE4K incorporated into POPC (1-palmitoyl-2-oleoyl-sn-phosphatidylcholine) liposomes was not measurably affected by pH, but increased membrane penetration was indicated by tryptophan fluorescence. At outer monolayer concentrations up to 10 mol%, Lipo-AcE4K formed stable liposomes with POPC and EPC/Chol (egg phosphatidylcholine/cholesterol) (55:45) at pH 7.5. Acid-induced destabilization and fusion of these vesicles were demonstrated by fluorescent lipid mixing and contents leakage assays, and by freeze-fracture electron microscopy. Membrane destabilization increased with increasing lipopeptide concentrations, decreasing pH, inclusion of cholesterol, and incorporation of lipopeptide into the inner monolayer as well as the outer monolayer of the liposomes. Fusion of liposomes bearing Lipo-AcE4K with erythrocyte ghosts was demonstrated by lipid mixing and fluorescence microscopy.

2D 1H-NMR conformational study of phosphatidylserine diluted in perdeuterated dodecylphosphocholine micelles. Evidence for a pH-induced conformational transition.

The conformation of phosphatidylserine (DMPS) diluted in perdeuterated dodecylphosphocholine micelles (DPC) has been investigated by 1D and 2D proton NMR spectroscopy. Chemical shift pH dependence showed that the pK relative to the serine carboxyl titration (3.4 +/- 0.05) was nearly identical to that measured in bilayers. Chemical shift and NOE data revealed that the phosphatidylserine molecule undergoes a conformational transition upon titration of the serine carboxyl group. The NOE network observed between the different parts of the molecule was sufficiently abundant to allow, in combination with molecular modeling methods, an assessment of the conformational changes. The conformational changes mainly involve the glycerol backbone, which is parallel to the whole molecule, that is, to the layer normal, at low pH and becomes perpendicular to the whole molecule at neutral pH. In both cases, the conformations are remarkably close to those observed for the crystal forms of zwitterionic and negatively charged phospholipids. Two-dimensional proton NMR study of phospholipids, diluted in perdeuterated DPC micelles, appears to be a simple and relevant method to obtain complete and direct information on their conformations in a model membrane-solution interface.

Studies of highly asymmetric mixed-chain diacyl phosphatidylcholines that form mixed-interdigitated gel phases: Fourier transform infrared and 2H NMR spectroscopic studies of hydrocarbon chain conformation and orientational order in the liquid-crystalline state.

Hydrocarbon chain conformational and orientational order in liquid-crystalline bilayers of the highly chain-asymmetric 1-O-eicosanoyl, 2-O-dodecanoyl and 1-O-decanoyl, 2-O-docosanoyl phosphatidylcholines were studied by Fourier transform infrared (FTIR) and deuterium nuclear magnetic resonance (2H-NMR) spectroscopy, respectively, and compared with appropriate symmetric-chain phosphatidylcholines at comparable reduced temperatures. FTIR spectroscopy indicates that these two asymmetric-chain phospholipids contain a slightly greater number of kink, a considerably larger number of double-gauche, but a somewhat smaller number of end-gauche conformers than does dipalmitoylphosphatidylcholine, a symmetric-chain phospholipid having the same total number of carbon atoms in its hydrocarbon chains. Moreover, the asymmetric-chain phospholipids also contain a larger total number of gauche conformers, suggesting that their hydrocarbon chains are more disordered overall than are those of dipalmitoylphosphatidylcholine. 2H-NMR studies of the specifically chain-perdeuterated analogs of these asymmetric-chain lipids reveal that the orientational order parameter profiles of their shorter and longer chains differ both qualitatively and quantitatively, regardless of whether they are esterified at the sn1- or sn2 positions of the glycerol molecule. The longer hydrocarbon chains exhibit unusual orientational order profiles in which the order gradient is steepest in the middle of the chain and relatively shallower in regions adjacent to the carboxyl and methyl termini, whereas the short hydrocarbon chains exhibit orientational order profiles typical of those commonly observed with conventional symmetric chain lipids. When compared at equivalent depths in the bilayer, the shorter hydrocarbon chains of the asymmetric-chain lipids are more orientationally disordered than are their longer chain counterparts. At comparable reduced temperatures, the shorter and longer chains of the asymmetric-chain lipids are more orientationally disordered than those of appropriate short and long symmetric-chain lipids, but the chain-averaged orientational order of the symmetric-chain lipid decreases more sharply with increases in temperature than does that of the comparable chain of the asymmetric-chain species. Moreover, the order plateau regions adjacent to the carboxyl groups of the longer chains of the asymmetric-chain phosphatidylcholines are shorter than those of symmetric-chain lipids of comparable hydrocarbon chain length. Overall, the data indicate that the conformational and orientational order in the liquid-crystalline states of these highly asymmetric-chain lipids differ significantly from those of comparable symmetric-chain lipids. Also, the unusual shape of the orientational order profile of the longer chains of the former is attributed to interaction between the methyl termini regions of the long chains with hydrocarbon chains in opposing monolayers. The latter suggests that some form of hydrocarbon chain interdigitation exists in liquid-crystalline bilayers of these highly asymmetric-chain lipids.

Evidence for two pools of cholesterol in the Acholeplasma laidlawii strain B membrane: a deuterium NMR and DSC study.

Recent investigations have indicated that there exists a well-defined range of membrane hydrocarbon order compatible with good growth of the microorganism Acholeplasma laidlawii B [Monck, M., Bloom, M., Lafleur, M., Lewis, R. N. A. H., McElhaney, R. N., & Cullis, P. R. (1992) Biochemistry 31, 10037-10043]. Since cholesterol increases hydrocarbon order in membranes, it was of interest to examine the effect of cholesterol on the hydrocarbon order and growth characteristics of A. laidlawii B. Cholesterol is normally absent from A. laidlawii membranes since it is neither biosynthesized nor required for the growth or survival of the microorganism. However, cholesterol will be incorporated into the membrane if exogenously supplied to the A. laidlawii culture. For membranes prepared from cells grown in the presence of cholesterol, chemical determinations indicated cholesterol represented as much as 40 mol% of the total membrane lipid. However, 2H NMR order parameter measurements and DSC studies of the same membrane preparation suggested that cholesterol was present at significantly lower levels (approximately 10-15 mol%) in the membrane lipid bilayer. Further incorporation of cholesterol into the A. laidlawii lipid bilayer was found to occur with an increase in temperature or by lyophilization and rehydration at high temperatures, suggesting that sterol present in a separate pool in the membrane preparation could then gain access to the bilayer. 2H NMR spectra of A. laidlawii membrane preparations containing deuterium-labeled cholesterol indicate that the bulk of the cholesterol present in this separate pool is in a solid form.

Influence of lipid composition on the orientational order in Acholeplasma laidlawii strain B membranes: a deuterium NMR study.

2H NMR techniques have recently been developed to determine the complete orientational order profile of lipid bilayers employing lipids containing perdeuteriated palmitic acid [Lafleur, M., Fine, B., Sternin, E., Cullis, P.R., & Bloom, M. (1989) Biophys. J. 56, 1037-1041]. In this work, these techniques have been applied to study order profiles in intact membranes derived from Acholeplasma laidlawii strain B. It is shown that complete orientational order profiles can be readily obtained from the intact membranes of A. laidlawii B grown on equimolar amounts of perdeuteriated palmitic acid and a nondeuteriated fatty acid of varying length and unsaturation. By variation of the fatty acid composition employing mixtures of perdeuteriated palmitic acid with myristic, elaidic, oleic, or linoleic acid, a range of hydrocarbon order compatible with high rates and extents of cell growth has been obtained where the average order parameter, mean value of S, varies over the range 0.140-0.176. This same variation in order is seen for liposomes derived from total lipids extracted from these intact membranes. 2H NMR studies on liposomes composed of individual species of the extracted lipids indicate that modulation of the membrane lipid headgroup composition has the potential to play an important role in maintaining the membrane order within this range.