RESUMEN
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
Mycobacterium tuberculosis infection continues to be a daunting clinical challenge. Although it may well be one of the most studied bacteria in history, several aspects of its pathology remain a mystery. The resurgence of drug-resistant M. tuberculosis strains and with its unusual pathology have promoted a renewed basic and clinical research interest in developing new therapies to combat this pathogen. The primary localization site for M. tuberculosis is within alveolar macrophages. Drug delivery strategies and novel therapeutic agents designed to target alveolar macrophages may lead to efficient destruction of M. tuberculosis. Oligodeoxynucleotides (ODN) are short segments of nucleic acids that can interfere with transcription and translation processes. In this report, a monocyte-macrophage cell line was characterized in regard to ODN transport in the presence or absence of M. tuberculosis infection. The cells accumulated ODN in a time-dependent and concentration-dependent manner, regardless of the presence of serum. After 4 hours of incubation with M. tuberculosis (multiplicity of infection [MOI] 10:1), infected NR8383 cells demonstrated 1.5-7-fold increase in fluorescein isothiocyanate (FITC)-labeled phosphorothioate ODN accumulation as measured by flow cytometry. The increase in uptake was associated only with fluorescent-labeled ODN and not labeled markers of fluid phase endocytosis (e.g., tetramethylrhodamine isothiocyanate [TRITC], FITC-labeled dextran). NR8383 cells activated by phytohemagglutinin (PHA) did not demonstrate a significant increase in the uptake of either FITC-labeled dextran or FITC-labeled ODN. These studies demonstrate that NR8383 cells that have been infected with M. tuberculosis can specifically accumulate ODN, and this route of accumulation may lead to a means of drug targeting to mycobacteria-containing cells.
Asunto(s)
Macrófagos Alveolares/microbiología , Mycobacterium tuberculosis/patogenicidad , Oligodesoxirribonucleótidos/metabolismo , Animales , Transporte Biológico , Línea Celular , Dextranos , Endocitosis , Citometría de Flujo , Fluoresceína-5-Isotiocianato/análogos & derivados , Infecciones por Mycobacterium/genética , Fitohemaglutininas/farmacología , RatasRESUMEN
The use of synthetic oligonucleotides (ONs) to systematically address new pharmacologic targets in mycobacteria would enhance the introduction of new molecular targets for drug intervention. Oligonucleotides' mechanism of action allows researchers to pursue the importance of particular proteins without the requirement of having purified samples. For this approach to be effective, mycobacteria must be able to transport ONs to their cytoplasm, and if this is not the case, the agents must be otherwise delivered. In this report, we characterize the ability of phosphorothioate (PS) and phosphorodiester (PD) ONs to interact with both Mycobacterium smegmatis and Mycobacterium tuberculosis. In addition, the use of delivery enhancer compounds, ethambutol and PAMAM dendrimer, was evaluated on the ON-mycobacteria interaction. ON interaction was demonstrated to be concentration-dependent, suggesting a possibly active component of the oligonucleotide and bacteria interaction. ON interaction could be increased by the coincubation of the bacteria with the delivery adjuvants. Treatment with ethambutol or dendrimers (fourth generation) was demonstrated to increase ON interaction with both species of mycobacteria although not to the same extent. The results of these preliminary experiments indicate that through use of the proper delivery adjuvant, ON interactions with mycobacteria can be increased. These findings may have implications for probing future antimycobacterial therapeutic targets.