Seite - 26 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Condeetal. Biofunctionalizationandsurfacechemistryof inorganicnanoparticles FIGURE14 |Biotin-avidinsystem.Schematicof inhibitionassaymethod basedon thephotoluminescencequenchingofSA-QDsbyBiotin-AuNPs. SAdenotes thestreptavidin immobilizedon thesurfaceofQDs,andAv is theexternally addedavidin (Fengetal., 2013).Reproducedwithpermission fromFengetal. (2013),Copyright2013. (Kd around 10−14) hasmade it perfect for the development of NPs-basedbiosensors.Themainmethodofbioconjugationusing avidin-biotin chemistry comprises the functionalization of NPs with avidin, for later incubation with a biotinylated molecule. Since avidin and its variants are zwitterionic molecules, they can be subject to electrostatic adsorption to negatively charged nanoparticles (see section Non-Covalent Strategies: Physical Interactions).Thevastnumberofpublications thatapplybiotin- avidin interaction for bioconjugation shows the importance of this strategy. Recently, Feng et al. showed that DNA detec- tion could be improved using streptavidin coated AuNPs (Feng et al., 2013). Another interesting strategy was reported by Oh et al. where by modulating the FRET efficiency between QDs and AuNPs they were able to detect molecules which inhibited the interactionbetween streptavidin andbiotin (Figure14) (Oh et al., 2005). By capping theAuNPswith polyamidoamine den- drimers, thebiotinylationwaspossibleusingsulfo-NHS-biotin. Asimilar strategywasemployed inthebiotinylationofMNPs. ToachieveaMNPs-PEG-biotinconjugatetheNPswereincubated with a phospholipid-PEG-biotin construct. By coupling DC14:0 PE (dimyristoylphosphatidylethanolamine) to further activate α-biotinylamido-ω-N-hydroxy-succinimidcarbonyl-PEG, the authors could produce MNPs covered with PEG-biotin. The functionalization was confirmed when binding streptavidin alkaline phosphatase the complexes became highly aggregated (Hodeniusetal., 2012). The strong association between avidin and biotin has made this system a reference for the development and troubleshoot- ing of NPs-based biosensors. It is also a crutch for conjugation of other biomolecules onto the surface of NPs. However, it is important to note that avidin is a glycoproteinwith a high iso- electric point (∼10). This could cause the unspecific binding of othercompoundspresentincomplexbiologicalsamples.Toover- come this problem, it is preferred theuseof streptavidin.As it is purified fromabacteria (Streptomycesavidinii) isnotaglycopro- tein andhasmuch lower isoelectricpoint (around5–6).Besides, the tetramericnatureof each(strept) avidinmoleculebecomesa problemwhencontroloftheAbstoichiometryisneeded.Toover- take this problem, it is possible touse recombinantmonomerics forms of these proteins but taking into account that the affin- ity for biotinwouldbemuch lower (around10−7M) (Wuet al., 2009b). CONCLUSIONSANDFUTUREPERSPECTIVES In the near future, it is expected that the design of nanosystems will revolutionizethemedicalhealthcarefieldbytheirapplication in the development of ultrasensitive andmultiplexed diagnostic systems, targeted and remotely controlled drug delivery systems for treatmentofdiseases, in vivo imaging, tissue/organ regenera- tionandgenetherapysolutions.The last threedecadeshavebeen anexcitingperiodinthesynthesisof inorganicnanoparticleswith interesting intrinsic properties for their use in suchapplications. Indeed,manyof these synthetic processeshavenotonlydemon- strated proof-of-concept feasibility but progressed to full-scale commercial production. However, optimization of appropriate sizescaleandbatch-to-batchreproduciblesyntheticproceduresof NPswithunique optical ormagnetic properties is not sufficient to ensure biomedical application. For this, functionalization of theNPswithbiomolecules iscrucial inorder to impartbiological recognitionandinteractionskills. Selectingthemostadequatebiofunctionalizationstrategyisno mean feat, since no universal methodologies exist to cover the wide variety of inorganic nanoparticles and biomolecules avail- ableforthispurpose.Afunctionalizationprotocolthatworkswell for one type ofNPmay notwork for another, since they could be very different in terms of size, charge, surface area, colloidal stability, density and type of reactive groups, etc. Furthermore, biomoleculesvarysignificantly in termsofsize, chemicalcompo- sition, 3Dcomplexity and locationof itsbiological active site.As discussedalong this review, inabsenceof standard functionaliza- tionprotocols,eachparticularcase(nanoparticle+biomolecule) requires optimization. Thus, in addition to the development of “smart”multifunctionalization strategies, it is vital to focus on thesynthesisof“smart”nanoparticlesoverthenextdecade.These NPs shouldbe able todeliver a therapeutic agent basedonenvi- ronmental causes or remote stimulus andwith the capability to temporarily adapt their size, shape, surface chemistry, wettabil- ity and adhesive properties to surrounding environments. These long-term goals would allow an overall impact on themedical fieldwith significant advances in patient screening,monitoring, diagnosis, staging,andtreatment. ACKNOWLEDGMENTS João Conde acknowledgesMarie Curie International Outgoing Fellowship (FP7-PEOPLE-2013-IOF, Project no. 626386). Pedro V. Baptista thanks CIGMH/FCT/MCES (PEst- OE/SAU/UI0009/2011-14). Jesus M. de la Fuente thanks MAT2011-26851-C02-01, Fondo Social Europeo, ERC-Starting Grant 239931-NANOPUZZLE, CDTI-INMUNOSWING, Shanghai100PeoplePlanandARAIDforfinancial support. 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