Efficient transfection of phosphorothioateoligodeoxyribonucleotidesby lipofectamine2000 into different bacteria.
Curr Drug Deliv. 2015 Aug 17;
Authors: Chen Z, Hu Y, Meng J, Li M, Hou Z, Zhou Y, Luo X, Xue X
Antisense technology has been a promising strategy for combating infectious diseases caused by multi-drug resistant bacterial strains, but the poor cellular uptake and transfection efficiency of these "antisense antibiotics" is strangling the development of antisense RNA therapeutics. This study was aimed at evaluating the cellular uptake characteristics and transfection efficiency of antisense phosphorothioateoligodeoxyribonucleotides (PS-ODN) in bacterial cells mediated by LipofectamineTM 2000 (LF2000). The size and surface morphology of LF2000/ODN nanoparticle were determined by dynamic light scattering and transmission electron microscope. Then the characteristics of cellular uptake was studied by flow cytometry analysis, and antibacterial efficacy of LF2000/ODN nanoparticle targeting rpoD, an RNA polymerase primary σ70, was tested by growth curve assay and RT-PCR. And the results showed the size of the spherical nanoparticle obtained was about 120 nm with a zeta potential about -5 mV, and the encapsulation efficiency of PS-ODN was about 95%. The cellular uptake efficiencies of LF2000/ODN nanoparticle by extended-spectrum β-lactamase-producing Escherichia coli (ESBLs-E. coli) and E. coli were 40.1% and 48.5% in a time-independent manner, while 76.7% and 79.3% by meticillin-resistant Staphylococcus aureus(MRSA) and S. aureusin a time-dependent manner. Interestingly, the uptake process was not impacted by the incubation temperature. After being incubated with LF2000/ODN, the growth of tested bacteria were significantly retarded, and the transcription of rpoDwas inhibited. Our research not only provided an experimental basis for further studies on delivery systems of antisense antibiotics, but also hinted a novel cellular uptake mechanism of nanoparticle.
PMID: 26279118 [PubMed - as supplied by publisher]