Branched peptide/DOTAP hybrid systems are efficient antisense oligonucleotide transfection reagents in bacteria
Xiaoyan Xue, Zhou Chen, Yu Hu, Ying Zhou, Zheng Hou, Mingkai Li and Xiaoxing Luo
The Fourth Military Medical University School of Pharmacy, China
: J Pharm Drug Deliv
Abstract
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 branched peptide/DOTAP hybrid system. The size and surface morphology of peptide/DOTAP/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 peptide/DOTAP/ODN nanoparticle targeting rpoD, an RNA polymerase primary Ã70, 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 -10 mV, and the encapsulation efficiency of PS-ODN was about 95%. The peptide/DOTAP/ODN nanoparticle could be efficiently uptaked by both Gram-positive bacteria and Gramnegative bacteria, and both drug-sensitive bacteria and drug-resistant bacteria, such as extended-spectrum β-lactamase-producing Escherichia coli (ESBLs-E. coli) and methicillin resistant Staphylococcus aureus (MRSA) in a time-independent manner. Interestingly, the uptake process was not altered by the incubation temperature. After being incubated with peptide/DOTAP/ODN, the growth of tested bacteria was 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.
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