Seite - 25 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Condeetal. Biofunctionalizationandsurfacechemistryof inorganicnanoparticles FIGURE12 | Ioniccoupling.Couplingofnegatively chargedsiRNA (left) andofpositively chargedproteins (right) tonegativelyorpositively charged AuNPs. Other examples of biological applications are the coupling of negatively charged DNA (Thomas and Klibanov, 2003; Ghosh et al., 2008;Conde et al., 2012b) or siRNA(Elbakry et al., 2009; Guo et al., 2010;Conde et al., 2012a; Zhao et al., 2012a) topos- itively charged NPs (Figure12) (Huschka et al., 2012). In fact, Conde et al. reported functionalization of siRNA by ionic cou- pling to apositively charged layer formedbyquaternary ammo- niumgroups (R4N+). Ionic interactions between the negatively chargedsiRNAbackbone(viaphosphategroups)andquaternary ammoniumpositively chargedgroups ensuredbindingof siRNA onto the AuNPs’ surface for the whole pH range. Using a hier- archicalapproachincludingthreebiologicalsystemsof increasing complexity, invitroculturedhumancells, invivo freshwaterpolyp (Hydra vulgaris) and in vivo healthymicemodel, these authors identifiedthemostadequatenanoparticlestoefficientlytransport siRNAs.Theresults evidencedthe importanceofacorrectdesign in the functionalization of nanoparticles for biological applica- tions, inparticular forcomplexanimalsystems,suchasmice.The ionic linkage of siRNAon the AuNPs showed efficiency in cells and inHydra. However, only a covalent bond ensured an active and efficient RNAi mechanism in mice (Conde et al., 2012a). Similarly, Li et al. reported the development ofQDs-DNAcom- plexes thataredisruptedandDNAreleasedbyglutathione(GSH) at intracellularconcentrations(Lietal., 2008). As extensively reviewed by others (Montenegro et al., 2013), conjugation of Abs to NPsmay bemade with covalent immo- bilization techniques such as those mentioned before, but also withnon-covalent strategies.Themostcommontechnique is the electrostatic adsorptionofAbsby charge interaction toopposite chargedNPs. This ionic adsorption is directly related to theAbs isoelectric point, pHatwhich they are neutral. Since it depends mainly on the number of charged groups, theAbs immobilized regionwill bewhere the greatest number of charges are present (Jung et al., 2008). Thismethod, although of easy implementa- tion, shows several disadvantages.Themain concern is theweak pHdependent interactionbetween theAb andNP.Any changes to the pH and/or ionic strengthmay incur in desorption of the Absmolecules. Additionally, the heterogeneous charge distribu- tionandtheunexploitedchargedgroupsof theAbscanpromote non-specificadsorption tomatrixproteins, for examplecompet- itivedisplacementcausedbyserumproteins(vanderVoortetal., 2004;MurciaandNaumann,2005). Hydrophobic coupling. Hydrophobic interactions have been widely exploited to attach lipophilic drugs to NPs, fromwhere FIGURE13 |Hydrophobiccoupling.Useofamphiphilicpolymersas nanoparticleanddrugdeliverymoieties. thedrugmightbereleasedonce inside thecells (Wahajuddinand Arora, 2012). For instance, docetaxel hasbeeneasily adsorbed to theoleic acid layer that surroundedhydrophobicMNPsprior to their encapsulation inapolymericvesicle,providingacontrolled drugreleaseprofile foramonth(Lingetal.,2011).Encapsulation of drugs can also bedone adsorbing them tohydrophobic poly- mers or cyclodextrins for instance (Yallapu et al., 2011) (see Figure13). Similarly, hydrophobic molecules such as fluorophores can be coupled toNPs. Foy and coworkers coated hydrophobicNPs withamphiphilicpolymers, towhich theyadsorbedfivedifferent dyes todetermineMNPbiodistributionusingamousexenograft breast tumor model (Foy et al., 2010). Moreover, Kim et al. developedAuNPs functionalizedwithwater-soluble zwitterionic ligandsfromkineticallystablecomplexeswithhydrophobicdrugs and dyes, which are efficiently released into cells (Kim et al., 2009). This couplingmethodminimizes changes to the surface andallowsthecreationofNPwithdualproperties,suchasoptical andtherapeutic, inaneasyway. Proteins andAbs can also be adsorbed toNPs viahydropho- bic interactions. However, they often suffer from denaturation, leading to poor reproducibility. Hydrophobic interactions with the hydrophobic surfaces of proteins or Abs force a change in the native structure because of exposure of its inner region, whichcouldultimately results in lossofactivity (Zuoetal., 2010; Shemetov et al., 2012).Moreover, regarding ionic binding, con- trolling the orientation or the amount of bound molecules is difficult toachieve. Biotin-avidin system.ForNPsconjugation, factors suchas solu- bility,chargeandall theaforementionedfunctionalgroupsconfer thebiotinarelevantimportance(Aslanetal.,2004).Biotinylation of NPs and biological molecules nowadays is fairly common as biotin can be synthesized to have a distal amine, thiol, car- boxyl and other functional groups, simplifying the conjugation (Hermanson, 2008). However, to avoid a random immobiliza- tion of structural complex biomolecules such as proteins, it is compulsory to achieve a site specific biotinilation. Concerning antibodies, for example, biotinilationmustbecarriedoutwithin itsFcregionvia thecarbohydratesmoietiesorvia thiolsobtained after reductionof thedisulfides located in thehinge region(Cho etal., 2007). Genetic engineering has also improved biotinylation by recombinantly introducing biotin labeling sites into fusion pro- teins (Cronan, 1990; Cull and Schatz, 2000). Nowadays several companies offer biomolecules andNPsmodifiedwith biotin or avidin species. The high-affinity of the avidin-biotin interaction Frontiers inChemistry | ChemicalEngineering July2014 |Volume2 |Article48 | 25