Seite - 23 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Condeetal. Biofunctionalizationandsurfacechemistryof inorganicnanoparticles FIGURE10 |Maleimidecouplingreaction.Maleimide reactswith free sulfhydryl group(s), formingstable thioether linkages, atphysiological pH. It isuseful forbioconjugationofproteinswith−SHgroupsand thecoupling of two thiols to formadisulfide linkage. applicationforfunctionalizationofothernanostructuredmateri- als, such as gold nanoprisms and carbonnanotubes (Polo et al., 2013). Maleimide coupling. Maleimide can be used to conjugate primary amines to thiols (Brinkley, 1992) (see Figure10). The use of maleimide for modification of sulfhydryl groups has been extensively described in the literature (Means and Feeney, 1990). Reactionwith sulfhydryl groups generates a sta- ble 3-thiosuccinimidyl ether linkage and occurs normally at pH 6.5–7.5. One of themain limitations is that themaleimide ring may hydrolyze in aqueous buffer to a non-reactive cis- maleamic acid derivative over long reaction times or at pH> 8.0.Nevertheless, this type of conjugation shows a lot of poten- tial for a great number of biomolecules that bear reactive thiol oraminogroups.Thismayeventually lead tonon-specificbonds and crosslinking between functionalized nanoparticles since a single biomolecule may have several thiol groups (Means and Feeney,1990;Brinkley,1992). Maleimide coupling has been used to conjugate several biomolecules toAuNPs, such as peptides (Oh et al., 2010a; Ravi et al., 2012), chemotherapeutic agents (Hwu et al., 2009), dyes (Zhu et al., 2012a), and DNA (Lee, 2011). In fact, Ba et al. presented a versatile and controlled route to immobilize AuNPs on the surface of living cells, while preserving the sensing and optothermal capabilities of the original colloid, by chemically anchoring the nanoparticles to phospholipids in liposomes via maleimide-thiol reactivity (Baetal., 2010). Maleimide couplingwas alsoused to coupleDNA(Dubertret et al., 2002), PNAs (Srinivasan et al., 2006), proteins (Wolcott etal.,2006;Bonasioetal.,2007;Zhouetal.,2007),andantibodies into QDs (Diagaradjane et al., 2008). To address biocompat- ibility issues of QDs, Dubertret et al. encapsulated individual nanocrystals in phospholipid block-copolymer micelles conju- gated to DNA and demonstrated their function as fluorescent probes (Dubertret et al., 2002). Bonasio et al. also reported the specific and covalent labeling ofQDswith amembrane protein andorganicfluorophores (Bonasioetal., 2007). Similarly, MNPs can also be functionalized using the maleimidecoupling reactionwithPEG(Kuhnetal., 2006),DNA (Nam et al., 2004) or even drugs, such as chlorotoxin (Kievit et al., 2010). Concerning antibodies, this chemistry could be also used with thiol or amino groups on the nanoparticle sur- face (Lee et al., 2007; Haun et al., 2010). Regarding a thiolated NP, antibodies would bind through their most reactive amine groups and, as previously explained, this could lead to a ran- domorientationwith partial loss of the Ab’s biological activity. Instead, maleimide chemistry used with aminated NPs ensures an oriented binding through thiol groups of the Ab. However, as in Abs sulfhydryls are oxidized as disulfides. So it is neces- sary to selectively reduce the disulfides at the hinge region by a reducing agent (i.e., 2-mercapthoethylamine, mercaptoethanol, dithiotreitol, thiopropyl-agarose). This chemical modification can also be combinedwith fragmentation of the IgGby the use of proteolytic enzymes (i.e., pepsine, ficin) inorder to conjugate smallAbfragments suchasF(ab′)2andFab′. Click-chemistry reaction. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction has been recognized as a facile and versatile chemistry for bioconjugation. Azides and alkynes are highly energetic functional groups with particularly narrowdistributionsofreactivity.Thanks totheirweakacid-base properties, they arenearly inert towardbiologicalmolecules and towardthereactionconditionsfoundinsidelivingcells.Theazide groups are easy to introduce into organic compounds by both nucleophilic and electrophilic processes. One of themost com- monbioconjugationofazidesisthecoppercatalyzedazide-alkyne cycloaddition (CuAAC)(Wanget al., 2003) (seeFigure11).This reaction features anenormous rate accelerationof107–108 com- pared to theuncatalyzed 1,3-dipolar cycloaddition (Himoet al., 2005).Thisreactionhasalsobeentermedthe“creamofthecrop” of click reactions and is surely responsible for the tremendous popularityofthe“click”conceptandmanysimplyassociate“click chemistry” to mean triazole formation between an azide and alkyne.Thereactionoccursat roomtemperature, showingahigh degreeof solventandpHinsensitivity, andhighchemoselectivity (theazideandalkyneare inert toreactwithnumerous functional groupsunder the typicallymild reactionconditions). In fact, the reaction succeeds over a broad temperature range, is insensitive to aqueous conditions and occurs in a pH range between 4 and 12 (Hein andFokin, 2010; Le et al., 2010).The copper catalyzed azide-alkyne cycloadditionoccurs between anorganic azide and a terminal acetylene.Thecyclicproduct is a triazole.The copper catalyst allows the reaction toproceed at room temperature and confersregioselectivity(areactioninwhichonedirectionofbond making or breaking occurs preferentially over all other possible directions),with the1,4regioisomerbeing theonlyproduct.The reactionstartbythe incubationwithamixtureofcopper(II)(e.g. copper(II) sulfate) and a reducing agent (e.g. sodiumascorbate) to produceCu(I) in situ (Meldal andTornoe, 2008;Hong et al., 2009). Click chemistry sometimes refers to a groupof reactions that are fast, simple to use, easy to purify, versatile, regiospecific, and give high product yields. However, the click reaction has a number of limitations. First, like with any cycloaddition, if the azide group is too electron deficient, then it will not undergo the reaction. In other words, the ground state configuration of the azide is far too low to interact with the terminal alkyne (Heinet al., 2008). Secondly, amore commonproblemis alkyne homocoupling. This phenomenonoccurswhen an alkyne reacts with a second alkyne instead of the azide. This process can be minimized by using a sterically bulky base that stabilizes the reactive intermediatesof thehomocoupling reactions.TheCu(I) saturationisrarebutcanalsobeaproblem,oncethealkynesmay Frontiers inChemistry | ChemicalEngineering July2014 |Volume2 |Article48 | 23