Jebali Ali, Anvari-Tafti Mohammad Hosssein
Department of Laboratory Sciences, School of Paramedicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Department of Pathobiology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
Colloids Surf B Biointerfaces. 2015 May 1;129:107-13. doi: 10.1016/j.colsurfb.2015.03.029. Epub 2015 Mar 18.
The use of antisense oligonucleotides is a novel strategy to treat infectious diseases. In this approach, vital mRNAs are targeted by antisense oligonucleotides. The aim of this study was to evaluate the effects of gold nanoparticles hybridized with different antisense oligonucleotides on Leishmania (L) major. In this project, gold nanoparticles were first synthesized, and then conjugated with primary oligonucleotides, 3'-AAA-5'. Next, conjugated gold nanoparticles (NP1) were separately hybridized with three types of antisense oligonucleotide from coding reign of GP63 gene (NP2), non-coding reign of GP63 gene (NP3), and both coding and non-coding reigns of GP63 (NP4). Then, 1mL of L. major suspension was separately added to 1mL of different hybridized gold nanoparticles at serial concentrations (1-200μg/mL), and incubated for 24, 48, and 72h at 37°C. Next, the uptake of each nanoparticle was separately measured by atomic absorption spectroscopy. After incubation, the cell viability was separately evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Also, the expression of GP63 gene was read out by quantitative-real-time PCR. This study showed that NP2 and NP3 had higher (5-fold) uptake than NP1 and NP4. Moreover, NP2 and NP3 led to less cell viability and gene expression, compared with NP1 and NP4. It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major. Importantly, these antisense oligonucleotides can be obtained from both coding and non-coding reign of GP63 gene. Moreover, hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L. major.
使用反义寡核苷酸是治疗传染病的一种新策略。在这种方法中，重要的信使核糖核酸（mRNA）被反义寡核苷酸靶向。本研究的目的是评估与不同反义寡核苷酸杂交的金纳米颗粒对硕大利什曼原虫（Leishmania (L) major）的影响。在这个项目中，首先合成金纳米颗粒，然后与初级寡核苷酸3'-AAA-5'共轭。接下来，将共轭金纳米颗粒（NP1）分别与来自GP63基因编码区（NP2）、GP63基因非编码区（NP3）以及GP63基因编码区和非编码区（NP4）的三种反义寡核苷酸杂交。然后，将1mL硕大利什曼原虫悬浮液分别加入到1mL不同系列浓度（1 - 200μg/mL）的杂交金纳米颗粒中，并在37°C下孵育24、48和72小时。接下来，通过原子吸收光谱法分别测量每种纳米颗粒的摄取量。孵育后，通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑（MTT）法分别评估细胞活力。此外，通过定量实时聚合酶链反应（qRT-PCR）读出GP63基因的表达。本研究表明，NP2和NP3的摄取量比NP1和NP4高（5倍）。此外，与NP1和NP4相比，NP2和NP3导致细胞活力和基因表达降低。可以得出结论，反义寡核苷酸的序列和大小对于硕大利什曼原虫的转染都很重要。重要的是，这些反义寡核苷酸可以从GP63基因的编码区和非编码区获得。此外，杂交金纳米颗粒不仅可以使GP63基因沉默，还可以杀死硕大利什曼原虫。
