RESUMO
Cholesterol 3-sulphate (CS) is a component of the intercellular lipid found in the uppermost layer of human epidermis (the 'stratum corneum') and is thought to play an important role in tissue cohesion. In this investigation we have compared the influence of cholesterol (CH) and CS on the gel-to-liquid crystalline phase behaviour, the polymorphic phase behaviour, and the hydrocarbon order profile in selected model membranes. It is shown that in sphingomyelin (SPM) systems, the presence of equimolar amounts of either CH or CS eliminates the gel-to-liquid crystalline transition as detected by calorimetry. Similarly, in 1-palmitoyl,2-oleoyl-phosphatidylethanolamine (POPE) dispersions containing a perdeuterated palmitoyl chain (POPE-d31), it is shown that both CH and CS exert an ordering effect as determined by 2H-NMR techniques, however, CS is less potent at temperatures both above and below that of the main transition for the native phospholipid. Alternatively, in mixed systems containing dioleoylphosphatidylethanolamine (DOPE) and SPM (DOPE/SPM, 6:1 mol/mol) CH promotes thermotropic L alpha-->HII phase transitions, whereas CS stabilizes the bilayer organization. These bilayer stabilization effects can be diminished by addition of Ca2+. These effects are consistent with a larger area per molecule of CS as compared to CH, presumably related to the presence of the negatively charged sulphate moiety of CS.
Assuntos
Membrana Celular/efeitos dos fármacos , Ésteres do Colesterol/farmacologia , Membrana Celular/química , Colesterol/farmacologia , Hidrocarbonetos/química , Bicamadas LipídicasRESUMO
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.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Bicamadas Lipídicas/química , Peptídeos/química , Fosfatidilcolinas/química , Sequência de Aminoácidos , Colesterol/química , Membrana Eritrocítica/química , Concentração de Íons de Hidrogênio , Lipoproteínas/química , Lipossomos , Fusão de Membrana , Dados de Sequência Molecular , Peptídeos/síntese química , Estrutura Secundária de Proteína , SolubilidadeRESUMO
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.
Assuntos
Carcinoma Pulmonar de Lewis/terapia , Terapia Genética , Lipídeos/administração & dosagem , Plasmídeos , Animais , Injeções Intravenosas , Taxa de Depuração Metabólica , Camundongos , Distribuição TecidualRESUMO
Human myelin basic protein (MBP) is shown to disrupt multilamellar phosphatidylcholine bilayers into small lipoprotein particles in a manner similar to the cytolytic peptide melittin (Dufourc, E. J., Smith, I. C. P., & Dufourcq, J. (1986) Biochemistry 25, 6448-6455). This bilayer fragmentation, as monitored by 31P nuclear magnetic resonance, is temperature-dependent and completely inhibited by the presence of small amounts of negatively charged phosphatidylserine. The stabilizing property of phosphatidylserine is lost with the neutralization of its negative charges upon membrane binding of cationic species such as calcium ions. No MBP-induced fragmentation is observed with bilayers of negative or zwitterionic lipid mixtures which mimic the myelin lipid composition. The membrane fragmentation observed in vitro in the presence of MBP could play a role in vivo in demyelinating diseases.
Assuntos
Bicamadas Lipídicas , Proteína Básica da Mielina/metabolismo , Cálcio/farmacologia , Humanos , Cinética , Proteína Básica da Mielina/isolamento & purificação , Fosfatidilcolinas , Fosfatidilserinas , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Deutério , Espectroscopia de Ressonância Magnética , Micelas , Fosfatidilserinas/química , Fosforilcolina/análogos & derivados , Glicerol/química , Concentração de Íons de Hidrogênio , Fosforilcolina/química , Conformação ProteicaRESUMO
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.
Assuntos
Fosfatidilcolinas/química , Deutério , Espectroscopia de Ressonância Magnética , Conformação Molecular , Fosfatidilcolinas/síntese química , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-AtividadeRESUMO
We report a new class of amphiphilic gemini surfactants as vehicles for gene delivery into cells, and the beginnings of a systematic structure-activity study. Preliminary results suggest that combining gemini surfactants with dioleoylphosphatidylethanolamine (DOPE) should allow the preparation of liposomes of various sizes and lipid compositions. Control of such colloidal changes could be as significant as the changes in the molecular composition of the gemini surfactants in delivering optimum gene expression in animal models.
Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Vetores Genéticos/química , Lipossomos/síntese química , Luciferases/metabolismo , Lipídeos de Membrana/química , Peptídeos/química , Peptídeos/síntese química , Fosfatidiletanolaminas/química , Tensoativos/química , Tensoativos/síntese química , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , DNA/metabolismo , Humanos , Luciferases/genética , Lisina/química , Espectroscopia de Ressonância Magnética , Camundongos , Microscopia Eletrônica , Estrutura Molecular , Músculos/citologia , Músculos/efeitos dos fármacos , Músculos/enzimologia , Neuroblastoma/enzimologia , Serina/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
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.
Assuntos
Acholeplasma laidlawii/metabolismo , Varredura Diferencial de Calorimetria , Colesterol/metabolismo , Espectroscopia de Ressonância Magnética , Membrana Celular/metabolismo , Deutério , Liofilização , Temperatura Alta , Bicamadas Lipídicas/metabolismo , TermodinâmicaRESUMO
Magnetic resonance spectroscopic (MRS) measurement of human plasma has been reported as a generally applicable marker for malignancy: patients with malignancy had a MRS line width significantly different from patients with benign diseases or healthy controls. The authors investigated the value of this test in 213 women with ovarian carcinoma, benign pelvic masses, benign nongynecologic diseases, and healthy controls. The MRS measurements were performed on plasma samples at 21 degrees C or 27 degrees C. The line width parameters were obtained by averaging the width at half the height of the methyl and methylene peaks on the resonance spectra. At 27 degrees C using 33 Hz as the threshold for an abnormal result, there was a significant correlation between the result of the test and the presence or absence of malignancy. However, the study demonstrates that the specificity (0.44) and positive predictive value (0.42) are too low for the test to be useful in the management of patients with carcinoma of the ovary. At 21 degrees C no correlation between the results of the test and the clinical status of women with carcinoma of the ovary were observed. In 47 patients the test did not predict preoperatively the benign or malignant nature of a pelvic mass.
Assuntos
Carcinoma/sangue , Neoplasias Ovarianas/sangue , Carcinoma/diagnóstico , Feminino , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Espectroscopia de Ressonância Magnética , Neoplasias Ovarianas/diagnóstico , Plasma/metabolismo , Valor Preditivo dos Testes , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Acholeplasma laidlawii/química , Membrana Celular/química , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/análise , Deutério , Ácidos Graxos/análise , Ácidos Graxos/química , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/química , Lipossomos/química , Lipídeos de Membrana/química , Ácido Palmítico , Ácidos Palmíticos/análise , Fosfatidilcolinas/química , Fosfatidilgliceróis/químicaRESUMO
The transfer of plasmid expression vectors to cells is essential for transfection after administration of lipid-based DNA formulations (lipoplexes). A murine i.p. B16/BL6 tumor model was used to characterize DNA delivery, liposomal lipid delivery, and gene transfer after regional (i.p.) administration of free plasmid DNA and DNA lipoplexes. DNA lipoplexes were prepared using cationic dioleoyldimethylammonium chloride/dioleoylphosphatidylethanolamine (50:50 mol ratio) liposomes mixed with plasmid DNA (1 microgram DNA/10 nmol lipid). The plasmid used contained the chloramphenicol acetyltransferase gene and chloramphenicol acetyltransferase expression (mU/g tumor) was measured to estimate transfection efficiency. Tumor-associated DNA and liposomal lipid levels were measured to estimate the efficiency of lipid-mediated DNA delivery to tumors. Plasmid DNA delivery was estimated using [3H]-labeled plasmid as a tracer, dot blot analysis, and/or Southern analysis. Liposomal lipid delivery was estimated using [14C]-dioleoylphosphatidylethanolamine as a liposomal lipid marker. Gene expression in the B16/BL6 tumors was highly variable, with values ranging from greater than 2,000 mU/g tumor to less than 100 mU/g tumor. There was a tendency to observe enhanced transfection in small (<250 mg) tumors. Approximately 18% of the injected dose of DNA was associated with these small tumors 2 h after i.p. administration. Southern analysis of extracted tumor DNA indicated that plasmid DNA associated with tumors was intact 24 h after administration. DNA and associated liposomal lipid are efficiently bound to tumors after regional administration; however, it is unclear whether delivery is sufficient to abet internalization and appropriate subcellular localization of the expression vector.
Assuntos
Técnicas de Transferência de Genes , Melanoma Experimental/metabolismo , Plasmídeos/administração & dosagem , Animais , Southern Blotting , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Escherichia coli/enzimologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Injeções Intraperitoneais , Lipídeos , Lipossomos , Melanoma Experimental/enzimologia , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Fosfatidiletanolaminas , Plasmídeos/genética , Plasmídeos/farmacocinética , Compostos de Amônio Quaternário , Distribuição TecidualRESUMO
The structure of 'stabilized plasmid-lipid particles' (SPLP) and their properties as systemic gene therapy vectors has been investigated. We show that SPLP can be visualized employing cryo-electron microscopy to be homogeneous particles of diameter 72 +/- 5 nm consisting of a lipid bilayer surrounding a core of plasmid DNA. It is also shown that SPLP exhibit long circulation lifetimes (circulation half-life >6 h) following intravenous (i.v.) injection in a murine tumor model resulting in accumulation of up to 3% of the total injected dose and concomitant reporter gene expression at a distal (hind flank) tumor site. In contrast, i v. injection of naked plasmid DNA or plasmid DNA-cationic liposome complexes did not result in significant plasmid delivery to the tumor site or gene expression at that site. Furthermore, it is shown that high doses of SPLP corresponding to 175 microg plasmid per mouse are nontoxic as assayed by monitoring serum enzyme levels, whereas i.v. injection of complexes give rise to significant toxicity at dose levels above 20 microg plasmid per mouse. It is concluded that SPLP exhibit properties consistent with potential utility as a nontoxic systemic gene therapy vector.
Assuntos
Terapia Genética/métodos , Vetores Genéticos , Neoplasias Pulmonares/terapia , Neoplasias Experimentais/terapia , Animais , Microscopia Crioeletrônica , Feminino , Expressão Gênica , Injeções Intravenosas , Lipídeos , Luciferases/genética , Camundongos , Camundongos Endogâmicos C57BL , PlasmídeosRESUMO
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.
Assuntos
Terapia Genética/métodos , Metabolismo dos Lipídeos , Animais , Cátions/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Endocitose , Fusão de Membrana , Camundongos , Modelos Biológicos , Oligonucleotídeos/química , Fosfatidiletanolaminas/farmacologia , Plasmídeos/química , Fatores de TempoRESUMO
Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70,000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid-degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.