Page - 14 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Condeetal. Biofunctionalizationandsurfacechemistryof inorganicnanoparticles FIGURE4 |SilencingtheSilencerswithhairpin-DNA-AuNPs—Gold nanobeacons.(A)SpecificAu-nanobeaconsarecapableofintersectingmiRNA pathway, leading to recoveryofpreviouslydownregulatedgeneexpression whilesimultaneouslydiscriminatingcellswheresilencing isoccurring.The fluorescencesignalmayallowfor trackingcell internalizationandsub-cellular localization.TheAu-nanobeacons’potential foranti-cancer therapeuticsvia the silencingof thesilencers isdemonstratedbyblocking theendogenous microRNApathwayviaanAnti-miRAu-nanobeaconcomplementary to the maturemicroRNA-21 (miR-21), commonlyupregulated incancerphenotypes. (B,C)Au-nanobeaconssilencingofendogenoussilencers—silencingof miR-21.Confocal imaging (scalebar,10μm)shows internalizationof50nM (B)Anti-miRAu-nanobeacon50nMand (C)NonsenseAu-nanobeacon.Target (maturemiR-21) recognition leads tochangeofAnti-miRAu-nanobeacon conformation in thecytoplasmwithconcomitantfluorescencesignal (red, Cy3)encircling thecell nuclei (blue,DAPI) (Condeetal., 2013b).Reproduced withpermission fromCondeetal. (2013b),Copyright2013. with a fluorophore labeled hairpin-DNA are capable of effi- ciently silencing single gene expression, exogenous siRNA and anendogenousmiRNAwhileyieldingaquantifiablefluorescence signaldirectlyproportional to the levelof silencing. Inhibitionof gene expression was achieved with concomitant increase of the gold nanobeacons’ fluorescence that can be used to assess the silencingeffect (Rosaetal., 2012;Condeetal., 2013a,b,2014b). Moreover, AuNPs functionalized with ssDNA are also capa- ble of specifically hybridizing to a complementary target for the detection of a particular nucleic acid sequence in biological samples (Sato et al., 2003; Storhoff et al., 2004; Baptista et al., 2005; Thaxton et al., 2006). The impact of advances in these nanoparticle-based assays for specificdetectionof bioanalytes of clinical interest is particularly relevant inbiodiagnostics,making themideal candidates fordevelopingbiomarkerplatforms. However, nucleic acidmolecules are also capableof establish- ing ionic interaction ingold surface. Ithasbeenshownthatboth thiol-ssDNAanddsDNAstabilize goldnanoparticle dispersions, but possible non-specific interactions between the hydrophobic DNA bases and the gold surface promote interparticle inter- actions and cause aggregation within a short period of time (Cardenasetal.,2006).ThechargerepulsionamongDNAstrands and between DNA and AuNPs can be reduced by adding salt, reducingpHorbyusingnon-chargedpeptidenucleicacid(PNA) (Zhangetal., 2012b). Moreover,Condeet al. reported thedesignof twoapproaches (seeFigure5) for thebindingof siRNAmolecules tomultifunc- tionalAuNPs: (Figure5A) the bindingof thenegatively charged siRNA through ionic interactions to the modified gold surface (ionic approach) and (Figure5B) the use of thiolated siRNA for the binding to the nanoparticle through the strong interac- tion gold-thiol (covalent approach) (Conde et al., 2012a). The covalent approachwas evaluated in a lung cancermousemodel to evaluate the inflammatory response and therapeutic siRNA silencing via RGD-nanoparticles. This study reported the use of siRNA/RGDgold nanoparticles capable of targeting tumor cells in two lungcancerxenograftmousemodels, resulting insuccess- ful and significant c-Myc oncogene downregulation followed by tumor growth inhibition and prolonged survival of the animals (Condeetal., 2013c). Modulation of the physicochemical properties of the gold nanocarriers canbe easily achievedby adequate synthetic strate- gies and give AuNPs advantages over conventional detection methods currently used in clinical diagnostics and therapy. Simple and inexpensivemethodsbasedon thesebio-nanoprobes wereinitiallyappliedfordetectionofspecificDNAsequencesand arecurrentlybeingexpandedtoclinicaldiagnosis (Baptistaetal., 2008). QDs have also been functionalized with nucleic acids and extensively used as DNA sensors (Crut et al., 2005; Dubertret, 2005;Zhanget al., 2005a;Choiet al., 2009;Yeet al., 2009;Zhang andHu,2010). For in vitro test of targetDNA,QDsbasedFRET pairs turned out to be of great use. Themethod developed by Zhangetal.usingQDs-Cy5-labeledreporteroligonucleotidecon- jugates, capable of detecting low concentrations of DNA in a separation-free format. This system uses QDs linked to DNA probes to capture DNA targets and successfully detect a point mutation(Zhangetal., 2005a). Owing to their unique optical properties, QDs have also been applied for multiplex detection of analytes with single- molecule detection. Zhang et al. reported the use of a single QD-basednanosensorformultiplexdetectionofHIV-1andHIV- 2 at single-molecule level in ahomogeneous format (Zhang and Hu, 2010). These QD-based nanosensors have several advan- tagessuchasextremely lowsampleconsumption,highsensitivity, short analysis timeandhave thepotential tobeapplied for rapid www.frontiersin.org July2014 |Volume2 |Article48 | 14