Seite - 17 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Condeetal. Biofunctionalizationandsurfacechemistryof inorganicnanoparticles Ramanreportermolecules,whichserveasextrinsic labelsforeach typeofantibody(Nietal., 1999). More recently,Condeet al. developedahighly sensitiveprobe for in vivo tumor recognition with the capacity to target spe- cific cancer biomarkers such as EGFR on human cancer cells and xenograft tumormodels. The authors used∼90nmAuNPs capped by a Raman reporter, encapsulated and entrapped by larger polymers and a Food and drug Administration (FDA) antibody–drug conjugate—Cetuximab (Erbitux®). These smart SERS gold nanoantennas present a high Raman signal both in cancer cells and in mice bearing xenograft tumors and the Ramandetectionsignal isaccomplishedsimultaneouslybyexten- sive tumorgrowth inhibition inmice.This approachseems tobe an innovative and efficient theranostics system for both tumor detectionandtumorcell inhibitionatthesametime(Condeetal., 2014a). QD-Antibody conjugates have also been widely used for preparing bioconjugated QDs for in vitro bioassay applications (Goldman et al., 2002; Hua et al., 2006; Tan et al., 2007; East et al., 2011). In fact, Goldman et al. described the preparation andcharacterizationofbioinorganicconjugatesmadewithhighly luminescent semiconductorCdSe-ZnS core-shell QDs and anti- bodies for use in fluoroimmunoassays.QD-antibody conjugates were successfully used influoroimmunoassays for detectionof a proteintoxin(staphylococcalenterotoxinB)andasmallmolecule (2,4,6-trinitrotoluene)(Goldmanetal., 2002). Concerningmagnetic NPs, bioseparation is one of themain applicationsofMNP-Abconjugates. In fact,magnetic separation of red blood cells usingmagneticmicrospheres was reported as early as 1977 (Moldayet al., 1977).MNPsareused topurify and concentrate different types of analytes in complex samples, such ashormones inbiological samples, antibioticsorbacteria in food (Kuo et al., 2012; Svobodova et al., 2012; Xu et al., 2012). One of thebestknownsystems that employMNPs for separationand concentration is thebio-barcodetechnologyoriginallydescribed byNametal. (2003). In thiscase,Absspecific fora targetprotein are functionalized on the surface ofMNPs, by sandwiching the targetbetweentheseMNPsandanamplifierAuNPsthat is loaded withasecondaryAbandoligonucleotides.Whenthespecific tar- get is sandwiched between the MNP and the AuNP, magnetic separationof the complexedprobes allows for the concentration of the targetwithin the sample.Afterwards, theoligonucleotides are released and detected, giving rise to a substantial amplifica- tion of the signal, and therefore lowering the detection limit to attomolar concentrations (Goluch et al., 2006). In fact, the first point-of-carenano-enabledmedical diagnostic tool approvedby the FDA, known as Verigene System and commercialized by Nanosphere Inc., isbasedonthisbiosensingstrategy. MNPsconjugatedwithAbsarealsouseful forthedevelopment of anewclass of diagnostics nanosensors, calledmagnetic relax- ationswitches(MRS).MRShavethepotential toprovidesensitive andselectivedetectionofavarietyofmolecular interactionswith minimal or no sample preparation (Perez et al., 2002). These assaysexploitthefactthatwhenMNPsrecognizeandbindbiolog- ical targets, they cluster, changing the spin-spin relaxation times of water protons (T2). These changes in T2 between dispersed and aggregated states of theMNPs canbemonitoredbynuclear magneticresonance(NMR)(Minetal.,2012).Oneofthegreatest advantagesofthesebiosensors is thattheyemployradiofrequency radiationwhichpenetrates biological samples regardless of their opticalproperties, and therefore canbeused incomplex samples such as blood. Using this technology, Lee et al. reported the firstmicroNMRbiosensor,where tumor cells couldbedetected employingMNPs functionalizedwithAbs onmicroliter sample volumesand inmultiplexed format (Leeet al., 2008). Since then, thesensitivityofthesebiosensorshasbeengreatlyimprovedusing otherhighlymagneticMNPs(Leeet al., 2009), so thatmolecular profilingofcancercellsobtainedbyfine-needleaspiratesbiopsies within60min ispossiblenowadays.Usingdifferentmarkers, the authorsreported96%accuracyforestablishingacancerdiagnosis (Haunetal., 2011). Other proteins have also been successfully conjugated with NPs (Mattoussi et al., 2000; So et al., 2006a,b; Xia et al., 2008; Roullier et al., 2009).Mattoussi et al. first described the electro- static interactionsbetweennegatively charged lipoic acid capped QDs and a positively charged recombinant protein (Mattoussi et al., 2000). Prasuhn et al. also developed aQDprotein FRET- basedbiosensors used as caspase 3proteolytic andCa2+ sensors (seeFigure6) (Prasuhnetal., 2010). Similar to nucleic acids, proteins are known for their spe- cificbinding interactions andcanact togetherwith awide range of substrates and synthetic analogs. Consequently, highmolec- ular weight peptide ligands show potential for wide biological applications and for stabilization and biofunctionalization of nanocarriers. Enzymes Enzymes, as highly specialized protein catalysts, are commonly used inbiofunctionalizationdue to theirpotential inbiotechnol- ogy and biomedicine, because of the convenience in handling, ease of separation from the reactionmixture and reuse, as well as low product cost. The immobilization in NPs often reduces diffusion limitations and/or enhances the catalytic activityof the enzymes. An important focus of the research onAuNPs based biosen- sors is in enzyme electrodes. One recurrent example is glucose biosensors. Zhang et al. (2005b) described the assembly of a gold electrode modified via Au-S bond with AuNPs, where a cystaminemonolayer is chemisorbed, thus exposing an array of aminogroups.Theseare further reactedwithaldehydegroupsof periodate oxidized glucose oxidase via Scchiff base reaction. In this study, the NPs showed to act as conduction intermediates facilitating electron transfer,with little effect onenzymeactivity. Itwasalso shownthat the sensitivitywas improvedaswell as the affinity forglucose,hence lowering thedetection limits. In another study, MNPs were modified with N- phosphonomethyl iminodiacetic acid for immobilization of urease. Thus, the surface coating was conferred with carboxyl groups towhichurease hadbeen immobilized through carbodi- imidereaction(Sahooetal.,2011).TheadvantageofusingMNPs is the possibility of product isolation by a permanent magnet, thus reducing costs. The authors also reported that the thermal stabilityof theureasewas increased, showing thatMNPsmaybe apromisingmaterial for storageandenzymeimmobilization. Frontiers inChemistry | ChemicalEngineering July2014 |Volume2 |Article48 | 17