Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(γ-glutamic acid) complex as a carrier

• Published in: PloS one 2013-06, Vol.8 (6), p.e65540
• Main Authors: Geng, Yao, Lin, Dajie, Shao, Lijia, Yan, Feng, Ju, Huangxian
• Format: Article
• Language: English
• Subjects: Acids; Amino acids; Analytical chemistry; Apoptosis; Biology; Biomedical materials; Breast cancer; Cancer; Cell Death; Chemical engineering; Chemistry; Chitosan; Chitosan - chemistry; Cytoplasm; Deoxyribonucleic acid; Disease prevention; DNA; Efficiency; Electrophoretic Mobility Shift Assay; Flow Cytometry; Fluorescence; Gene expression; Gene transfer; Gene Transfer Techniques; Genetic vectors; Glutamate; Glutamic acid; Gold; Hostages; Humans; Hybridization; Hydrolases; Intracellular; Intracellular Space - metabolism; Investigations; Laboratories; Life sciences; Materials Science; MCF-7 Cells; Medical research; Methods; MicroRNA; MicroRNAs; MicroRNAs - genetics; MicroRNAs - metabolism; miRNA; Nanoparticles; Nucleic Acid Hybridization; pH effects; Polyglutamic Acid - analogs & derivatives; Polyglutamic Acid - chemistry; Quantum Dots; Real-Time Polymerase Chain Reaction; Ribonuclease; Ribonuclease III; Ribonucleic acid; RNA; RNA probes; RNA Probes - metabolism; Target detection; Transfection
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0065540

A quantum dot (QD)-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP) via Au-S bond and then binding 3'-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics.