RESUMEN
The ability of a cationic lipid to deliver plasmid DNA (pDNA) in presence of the neurotoxic fragment of amyloid beta-peptide was evaluated. Pre-treatment of cells with betaAP (25-35) peptide resulted in a modest increase in transgene expression. When betaAP (25-35) peptide was mixed with the pDNA/liposome complex and used, the complexes lost their ability to transfect. However, the reverse sequenced betaAP (35-25) peptide demonstrated no significant differences in transgene expression in pre-treated cells, and in cells where betaAP (35-25) peptide was mixed with pDNA/liposome complexes and transfected. The amount of pDNA delivered to the cells was decreased in presence of betaAP (25-35) as measured with flow cytometry using fluorescently labeled liposomes. The decreased endocytosis may be due to their rod-like structure formation as demonstrated by electron microscopy and atomic force microscopy (AFM). These results demonstrate that betaAP (25-35) peptide may interfere with gene delivery with cationic systems.
Asunto(s)
Péptidos beta-Amiloides/administración & dosificación , ADN/administración & dosificación , Técnicas de Transferencia de Gen , Lípidos/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Transgenes , Péptidos beta-Amiloides/metabolismo , Animales , Células Cultivadas , Endocitosis , Citometría de Flujo , Humanos , Microscopía Electrónica , Fragmentos de Péptidos/metabolismo , Plásmidos , Ratas , Células Tumorales CultivadasRESUMEN
The impact of biodegradable pH-sensitive surfactant (BPS)-liposomes on nucleic acid, i.e., oligonucleotide and plasmid DNA, cellular delivery was examined. Fluorescein-labeled nucleic acids complexed with 1,2-dioleoyl-3-trimethylammonium propane cationic liposomes and BPS at a charge ratio (+/-) of 10 were incubated in CV-1 cells and analyzed by flow cytometry. The fluorescence intensity of oligonucleotides but not plasmid DNA complexed with BPS-liposomes was higher than those complexed with BPS-free liposomes at early time points. However, when cells were fixed to equalize the intracellular pH since fluorescein, a pH-sensitive fluorophore, has higher fluorescence intensity in alkaline pH than acidic, no difference in intensity was observed. This indicated the incorporation of BPS in liposomes did not increase oligonucleotide cellular uptake over control liposomes, but redistributed oligonucleotides into a more basic environment, e.g., cytoplasm. An explanation consistent with the presented data is the formation of small transient membrane defects within the endosomal membrane as presented previously [Liang, E., Hughes, J.A., 1998a. Membrane fusion and rupture in liposomes: effect of biodegradable pH-sensitive surfactants. J. Membr. Biol. 166, 37-49.]. The above findings suggested that BPS may be effective agents of disrupting one of the major barriers, endosomal membrane, to enhance nucleic acid cellular transport.
Asunto(s)
Ácidos Grasos Monoinsaturados/farmacocinética , Oligonucleótidos/metabolismo , Plásmidos/metabolismo , Compuestos de Amonio Cuaternario/farmacocinética , Tensoactivos/farmacocinética , Animales , Biodegradación Ambiental , Línea Celular , Portadores de Fármacos , Fluoresceínas , Concentración de Iones de Hidrógeno , Liposomas , Distribución TisularRESUMEN
The use of oligonucleotides (ONs) for gene therapy of certain diseases has been discussed since the late 1970s. ONs are single stranded chains of nucleic acids that can hybridize with target nucleic acid sequences to inhibit specific proteins, and therefore allow selective treatment of various diseases. The use of ONs is limited due to their instability in biological tissues and difficulty in delivery to the intracellular compartments of the cell. Chemical analog approaches have been used to address the instability issue and delivery systems have been developed to increase cellular uptake of ONs. It is generally thought that ONs with or without a delivery system are transported into cells by endocytosis, and then accumulate within endosomes where they are significantly inactivated. The rate and extent of movement of ON from endosomes appears to be important in determining ON effects. Consequently, developing accessory compounds or delivery methods that enhance endosome to cytoplasm transfer may be vital to ON therapy. This review focuses on investigating mechanisms of various delivery approaches at the cellular/intracellular level that have demonstrated utility in increasing ON activity or cellular accumulation. The future prospects of ON delivery are also addressed.
Asunto(s)
Marcación de Gen/métodos , Terapia Genética/métodos , Oligonucleótidos/uso terapéutico , Animales , Humanos , Oligonucleótidos/administración & dosificaciónRESUMEN
Cationic lipid formulations consisting of 3beta [N-(N', N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the helper lipid dioleoylphosphatidylethanolamine (DOPE) (1.5: 1 molar ratio) were prepared by solvent evaporation and sized by high pressure extrusion. Liposomes made of 1:1 molar ratio 1 ,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP)/DOPE were used as controls in the study. The two formulations were characterized and evaluated for their efficiency in transfecting SKnSH (neuroblastoma) and primary rat neuronal cell lines. DC-Chol/DOPE liposomes were more efficient at transfecting both the SKnSH and the primary rat neuronal cells and also less toxic compared to the DOTAP/DOPE liposomes. The cellular-associated signal of rhodamine-labeled DC-Chol/DOPE liposomes into SKnSH and primary rat neuronal cells was higher than the rhodamine-labeled DOTAP/DOPE liposomes. These results demonstrate that DC-Chol/DOPE cationic liposomes provide an efficient vehicle for the delivery of plasmids into SKnSH and primary neuronal cells compared to DOTAP/DOPE liposomes. DC-Chol/DOPE liposomes may provide a good non-viral candidate for transfecting primary rat neuronal cells.
Asunto(s)
Colesterol/análogos & derivados , Técnicas de Transferencia de Gen , Liposomas , Neuronas/metabolismo , Animales , Citometría de Flujo , Colorantes Fluorescentes , Vectores Genéticos , Humanos , Neuroblastoma , Fosfatidiletanolaminas , Plásmidos/genética , Ratas , Rodaminas , Células Tumorales CultivadasRESUMEN
Deoxycholic acid-spermine conjugate (DAS), which is composed of natural components (deoxycholic acid and spermine), was incorporated in liposomes and evaluated for its interaction with plasmid DNA (pDNA) and in vitro transfection efficiency. Electromicrographs demonstrated that DAS-pDNA complexes are spherical, compact and electronically dense compared to the toroidal shapes formed by the monovalent lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and pDNA. In comparison to the singly charged, non-cholesterol based lipid (DOTAP), the multivalent lipid DAS had similar transfection efficiency in two cell lines. The monovalent sterol, deoxycholic acid propyldiamine conjugate (DAP) was not effective as a transfecting agent. This suggests that multivalent facial amphiphiles such as DAS may serve as excellent candidates for non-viral gene transfer and warrant further study.
Asunto(s)
Ácido Desoxicólico/química , Lípidos/química , Espermina/química , Transfección/genética , Animales , Células CHO , Cricetinae , ADN/química , ADN/genética , Portadores de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Ácidos Grasos Monoinsaturados/química , Liposomas , Microscopía Electrónica , Plásmidos/química , Plásmidos/genética , Compuestos de Amonio Cuaternario/química , Células Tumorales CultivadasRESUMEN
Several factors influence non-viral transfection in tissue culture models including nature of the cationic lipid, plasmid construction, and DNA lipid complex, among others. The cell line itself is another confounding variable. Each subcellular population may respond independently to the transgene or specific delivery vector with regards to toxicity or transgene expression. In this study, the SKnSH (human neuroblastoma) and COS-1 (African green kidney) cells were exposed to three different treatments A, B, and C. Treatment A refers to cells obtained from American Type Culture Collection (ATCC) and cultivated as recommended, treatment B to cells that were grown in presence of cationic lipids for two weeks, and treatment C to cells that were grown in presence of cationic lipids for two weeks followed by normal media for two weeks to determine if lipid mediated effects were reversible. Treatment B resulted in a three-fold increase in transgene expression of a reporter gene as compared to the other treatments. This increase in transgene expression appeared not to be related to alterations in toxicity. Interestingly, the fluid phase endocytic uptake of fluorescently labeled oligonucleotides was increased in treatment B. However, there was no significant difference in the cellular-associated signal when fluorescently labeled plasmid-DNA was evaluated. In COS-1 cells, no difference in transfection was observed with treatment B illustrating that cell lines respond independently. In conclusion, pre-exposure of SKnSH cells to cationic liposomes (treatment B) resulted in higher transgene production.
Asunto(s)
Marcación de Gen/métodos , Lípidos/farmacología , Transgenes , Animales , Células COS , Cationes , Línea Celular , Chlorocebus aethiops , Técnicas de Cultivo , ADN/administración & dosificación , ADN/genética , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Genes Reporteros/genética , Humanos , Lípidos/química , Neuroblastoma/metabolismo , Oligonucleótidos/metabolismo , Células Tumorales CultivadasRESUMEN
The transfection efficiency of a disulfide-containing cationic lipid, 1',2' dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO) and its non-disulfide analog (DOGSHDO) were compared in neuronal, astroglial and microglial cultures from newborn rat cerebral cortex. We hypothesized that the relatively high intracellular concentrations of reductive substances in the cytoplasm may help to cleave the reversible disulfide bond in DOGSDSO, thus increasing free DNA and decreasing toxicity due to rapid degradation of the lipid. We have demonstrated through mass spectrometric analysis that a reductive compound, e.g. dithiothreitol (DTT) could degrade the disulfide lipid. DOGSDSO was more efficient at transfecting each type of brain cell than were the non-disulfide DOGSHDO and DOTAP (1,2-dioleoyl-3-trimethyl-ammonium-propane) liposomes. These results demonstrate that disulfide-containing cationic liposomes facilitate gene transfection in cultured rat brain cells.
Asunto(s)
Corteza Cerebral/fisiología , Disulfuros/administración & dosificación , Expresión Génica/efectos de los fármacos , Transgenes/fisiología , Animales , Astrocitos/fisiología , Cationes/análisis , Células Cultivadas , Corteza Cerebral/citología , Disulfuros/farmacología , Portadores de Fármacos , Ácidos Grasos Monoinsaturados/administración & dosificación , Ácidos Grasos Monoinsaturados/farmacología , Lípidos/administración & dosificación , Lípidos/farmacología , Liposomas/química , Microglía/fisiología , Neuronas/fisiología , Ornitina/administración & dosificación , Ornitina/análogos & derivados , Ornitina/farmacología , Compuestos de Amonio Cuaternario/administración & dosificación , Compuestos de Amonio Cuaternario/farmacología , Ratas , Ratas Sprague-Dawley , Transfección/efectos de los fármacosRESUMEN
BACKGROUND: The ribonucleoprotein enzyme, telomerase, uses RNA as a template to add a hexanucleotide to ends of replicating chromosomes resulting in stabilization of the telomere. Telomerase activity is observed in over 85% of human primary malignancies, suggesting that it may be a new marker of cancer and raising the possibility that antitelomerase therapy may provide a new generation of cancer therapeutics. MATERIALS AND METHODS: In this study, we evaluated phosphorothioate (PS) oligonucleotide (ODNs) inhibition of telomerase activity in intact cells and cell lysates in the SKnSH (neuroblastoma) cell line. RESULTS: The ODNs were effective at inhibiting telomerase activity in cell lysate but demonstrated little effect when intact cells were used. CONCLUSIONS: This study demonstrates that cellular transport may be a limiting factor with therapeutic approaches using antisense ODNs.