Gold nanobeacon

Universal gene silencing tool based on gold nanoparticles functionalized with a fluorophore labeled hairpin-DNA – Gold nanobeacon (Au-nanobeacon) – that effectively detects and silences the specific target (siRNA, miRNA or gene specific mRNA) while simultaneously signalling its action via fluorescence emission in cancer cells.

Gold nanobeacons are gold nanoparticles functionalized with a fluorophore-labelled hairpin-DNA, i.e. gold-nanobeacon to follow RNA synthesis in real time in bulky solutions and for antisense DNA and RNA interference (RNAi), from gene specific silencing to silence-the-silencers. Under hairpin configuration, proximity to gold nanoparticles leads to fluorescence quenching; hybridization to a complementary target restores fluorescence emission due to the gold nanobeacons' conformational reorganization that causes the fluorophore and the gold nanoparticle to part from each other.


Theranostics

Gold nanoparticle-based molecular beacon have shown the capability to specifically detect DNA target sequences,[1] with better discriminating power for single-mismatch than regular molecular beacons.[2] Nevertheless, the systems described so far rely on the detection of nucleic acids in bulk solution and can only be used in in vitro applications without translation to cell/tissue/organism testing.

Recently, Conde et al. developed a new theranostic system capable of intersecting all RNA pathways: from gene specific downregulation to silencing the silencers, i.e. siRNA and miRNA pathways. The authors reported the development of new nanomaterials, i.e. gold nanoparticles functionalized with a fluorophore labeled hairpin-DNA - Gold nanobeacons - capable of efficiently silencing single gene expression, exogenous siRNA and endogenous miRNAs while yielding a quantifiable fluorescence signal directly proportional to the level of silencing.[3]

This method describes a gold nanoparticle-based nanobeacon as an innovative theranostic approach for detection and inhibition of sequence-specific DNA and RNA for in vitro and ex vivo applications.[4] This concept can easily be extended and adapted to assist the in vitro evaluation of silencing potential of a given sequence to be later used for ex vivo gene silencing and RNAi approaches, with the ability to monitor real-time gene delivery action.[5]

The mechanism proposed in the gold-nanobeacon tool has got several clear advantages compared with traditional methods. Firstly, naked/unmodified oligonucleotides show extremely short half-lives inside cellular environment, feeble protection against RNases and other nucleases, poor chemical stability, and common dissociation from vector. In fact, the major obstacle to clinical application is the uncertainty about how to deliver therapeutic DNAs (antisense oligonucleotides) and RNAs (e.g., microRNA and/or siRNA) with maximal therapeutic impact due to systemic loss of cargo from traditional vectors. This method offers an unprecedented opportunity to overcome these problems as these nanoconjugates can readily interact with biomolecules on both the surface of cells and inside cells for longer periods of time, due to their small size and protective environment for DNA/RNA oligonucleotides provided by the metal nanoparticle core. The gold-nanobeacons are also highly soluble, homogenous and stable and are not prone to aggregation. In addition, these nanoparticles are thermodynamically stable and can remain inside cells for long periods of time and at low concentrations. Secondly, gold-nanobeacons are capable of efficiently silencing single gene expression, exogenous siRNA and endogenous miRNAs.[6] Thirdly, it allows real-time detection of the beacon's signal while yielding a quantifiable fluorescence directly proportional to the level of gene silencing.[7] This can be used to track the silencing events inside the cell as they occur. All this is achieved in a simple approach that can be straightforwardly adapted and tailored to any specific target. Finally, a significant attribute of these gold-nanobeacons is the ability to attain similar levels of inhibition of gene expression with lower amounts than those of free oligonucleotides without increasing cell death. This extraordinary efficiency occurs probably due to the large payload capacity of the NPs and the longer half-life when inside the cells. The gold-nanobeacons may represent in the near future an economically viable and commercial-scale production for a cell and cell-free system.[8]

Contrary to conventional gene delivery chemistry, which is often associated to systemic toxicity and adverse effects as well as lack of specificity and lower product life-cycle, this method represents a safe, efficient, specific and non-pathogenic vehicle for gene delivery and cell tracking system – a theranostic tool.[9]

Bioresponsive antisense DNA gold nanobeacons for the inhibition of cancer cells and metastasis

Gold nanobeacons can be used as a tool for cancer theranostics. Recently, Conde et al. developed a nanomaterial platform based on gold nanobeacons to detect, target and inhibit the expression of a mutant Kras gene in an in vivo murine gastric cancer model.[10] The conjugation of fluorescently-labeled antisense DNA hairpin oligonucleotides to the surface of gold nanoparticles enables using their localized surface plasmon resonance properties to directly track the delivery to the primary gastric tumor and to lung metastatic sites. The fluorescently labeled nanobeacons reports on the interaction with the target as the fluorescent Cy3 signal is quenched by the gold nanoparticle and only emit light following conjugation to the Kras target owing to reorganization and opening of the nanobeacons, thus increasing the distance between the dye and the quencher. The systemic administration of the anti-Kras nanobeacons resulted in approximately 60% tumor size reduction and a 90% reduction in tumor vascularization. More important, the inhibition of the Kras gene expression in gastric tumors prevents the occurrence of metastasis to lung (80% reduction), increasing mice survival in more than 85%. The developed platform can be easily adjusted to hybridize with any specific target and provide facile diagnosis and treatment for neoplastic diseases.

2-pair FRET/NSET nanoswitch to sense, inhibit and deliver drugs

Now with one single local application using hydrogel scaffolds embedded with a 2-pair FRET/NSET (Fluorescence Resonance Energy Transfer/NanoSurface Energy Transfer) gold nanobeacons (also known as dark-gold nanobeacons) we are able to overcome drug resistance by detecting and silencing a multidrug resistance protein (MRP1), before chemotherapeutic drug delivery in vivo. Our platform contains hydrogel embedded with dark-gold nanoparticles modified with 5-fluorouracil (5-FU)-intercalated nanobeacons that serve as an ON/OFF molecular nanoswitch triggered by the increased MRP1 expression within the tumour tissue microenvironment.[11] This nanoswitch can sense and overcome multidrug resistance (MDR) prior to local drug release. These nanoprobes comprise a thiol-DNA-hairpin labelled with a NIR dye and a thiol-DNA oligo labelled with a dark quencher (BHQ2), polyethylene glycol (PEG), and intercalated drug- all of which are conjugated to a gold nanoparticle core. These dark-gold nanobeacons are then loaded with 5-FU that intercalates in the beacon stem (dsDNA part) of the DNA-hairpin oligo. Under hairpin configuration, the proximity of the NIR dye to the dark quencher leads to fluorescence quenching. Hybridization of the DNA hairpin to a complementary mRNA target restores fluorescence emission due to the gold nanobeacons' conformational reorganization that causes the fluorophore and the quencher to part from each other, yielding a quantitative response. On the other hand, the release of the 5-FU drug can only occur when DNA hairpin hybridizes with the complementary target and can be measured once the distance from the 5-FU and the gold core increases, escalating the drug emission. To evaluate the efficiency of the dark-gold nanobeacon probes in sensing and in overcoming MDR in vivo, an orthotopic breast cancer mouse model was developed by injecting 5-FU resistant MDAMB-231 cells to the mammary fat pad of female SCID hairless congenic mice. Efficacious and local delivery of the dark-gold nanobeacon probes is achieved by the implantation of a hydrogel disk on top of the triple-negative breast tumours using a polyamidoamine (PAMAM G5) dendrimer cross-linked with dextran aldehyde, which provides enhanced stability of the embedded nanoparticles. Despite the cross-resistance to 5-FU, more than 90% tumour reduction is achieved in vivo, following 80% MRP1 silencing compared with the continuous tumour growth following only drug or nonsense nanobeacon administration.

See also

References

  1. ↑ Song,S. et al. Gold-nanoparticle-based multicolor nanobeacons for sequence-specific DNA analysis. Angew. Chem. Int. Ed. Engl. 48, 8670-8674 (2009).
  2. ↑ Dubertret,B., Calame,M., & Libchaber,A.J. Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol. 19, 365-370 (2001).
  3. ↑ Conde J, Rosa J, de la Fuente JM, Baptista PV. Gold-nanobeacons for simultaneous gene specific silencing and intracellular tracking of the silencing events. Biomaterials. 2013;34(10):2516-23. doi: 10.1016/j.biomaterials.2012.12.015.
  4. ↑ Rosa J, Conde J, de la Fuente JM, Lima JC, Baptista PV. Gold-nanobeacons for real-time monitoring of RNA synthesis. Biosens Bioelectron. 2012;36(1):161-7. doi: 10.1016/j.bios.2012.04.006.
  5. ↑ Conde J, Rosa J, Baptista P. Gold-Nanobeacons as a theranostic system for the detection and inhibition of specific genes. Community Contributed Protocol Exchange. 27/11/2013. doi:10.1038/protex.2013.088.
  6. ↑ Conde J, Rosa J, de la Fuente JM, Baptista PV. Gold-nanobeacons for simultaneous gene specific silencing and intracellular tracking of the silencing events. Biomaterials. 2013;34(10):2516-23. doi: 10.1016/j.biomaterials.2012.12.015.
  7. ↑ Rosa J, Conde J, de la Fuente JM, Lima JC, Baptista PV. Gold-nanobeacons for real-time monitoring of RNA synthesis. Biosens Bioelectron. 2012;36(1):161-7. doi: 10.1016/j.bios.2012.04.006.
  8. ↑ Conde J, Rosa J, Baptista P. Gold-Nanobeacons as a theranostic system for the detection and inhibition of specific genes. Community Contributed Protocol Exchange. 27/11/2013. doi:10.1038/protex.2013.088.
  9. ↑ Conde J, Larguinho M, Cordeiro A, Raposo LR, Costa PM, Santos S, Diniz MS, Fernandes AR, Baptista PV. Gold-nanobeacons for gene therapy: evaluation of genotoxicity, cell toxicity and proteome profiling analysis. Nanotoxicology.(2013).
  10. ↑ Bao C, Conde J, Curtin J, Artzi N, Tian F, Cui D. Bioresponsive antisense DNA gold nanobeacons as a hybrid in vivo theranostics platform for the inhibition of cancer cells and metastasis. Scientific Reports 2015 Jul 20;5:12297. doi: 10.1038/srep12297.
  11. ↑ Conde J, Oliva N, Artzi N. Implantable hydrogel embedded dark-gold nanoswitch as a theranostic probe to sense and overcome cancer multidrug resistance. PNAS 2015;112(11):E1278–E1287. .

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