Efficient Transfection of Phosphorothioate Oligodeoxyribonucleotides by lipofectamine2000 into Different Bacteria.
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 phosphorothioate oligodeoxyribonucleotides (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 were studied by flow cytometry analysis, and antibacterial efficacy of LF2000/ODN nanoparticle targeting rpoD, an RNA polymerase primary σ70, was tested by analyzing the growth inhibition of targeted bacteria and by RT-PCR analysis of the target genes. And the results indicated that 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. aureus in a time-dependent manner. Interestingly, the uptake process was not altered by the incubation temperature. After being incubated with LF2000/ODN, the growth of tested bacteria were significantly retarded and the transcription of rpoD was inhibited. Our research not only provided a basis for further studies on delivery systems for antisense antibiotics, but also highlighted a novel cellular uptake mechanism of nanoparticle.
Zhou
Z
Chen
Yue
Y
Hu
Jingru
J
Meng
Mingkai
M
Li
Zheng
Z
Hou
Ying
Y
Zhou
Xiaoxing
X
Luo
Department of Pharmacology, School of Pharmacy, The Fourth Military Medical University, No.169, Changle West Road, Xi`an 710032, China. xxy.0707@163.com.
Xiaoyan
X
Xue
D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D008027:Light; D008055:Lipids / Q000737:chemistry*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D046529:Microscopy, Electron, Transmission; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010316:Particle Size; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D012542:Scattering, Radiation; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*
D008055:Lipids; C086724:Lipofectamine; D009838:Oligodeoxyribonucleotides; D054735:Phosphorothioate Oligonucleotides; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases