Seite - 11 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Condeetal. Biofunctionalizationandsurfacechemistryof inorganicnanoparticles markers, enzymes and other proteins that will introduce the requiredbio-functionalities. Ultimately, theconjugationstrategy isdirectlydependentona numbers of factors such as size, surface chemistry and shape, as well as the typeof ligandsandfunctionalgroups toexploit in the functionalization. Also, the type of biologicalmolecule and the final application of the nanoparticle conjugate are crucial when evaluatingtheconjugationstrategy.Next,wesummarizethemost frequentlyusedbiofunctionalmoleculesusedtointroduceoneor severalbiologicalactivities to theNP. COMMONBIOFUNCTIONALSPECIES Polymercoatings—poly(ethyleneglycol) For their use as potential delivery devices in vivo, the aforemen- tioned inorganicnanoparticlesmusthave longplasmahalf-lives. In this sense,poly(ethyleneglycol) (PEG) is themostwidelyused macromolecule to prolong nanocarriers half-life. In fact, PEGs have a strong effect on nanoparticle structure, stabilization and biodistribution both in vitro and in vivo (Akerman et al., 2002; Daou et al., 2009; Boenemanet al., 2010;Maldiney et al., 2011). These long-circulatingnanoparticles have the ability to circulate for a prolonged period of time and target a particular organ, as carriers ofDNA in gene therapy, or to deliver proteins, peptides anddrugs (Langer,2000;Bhadraetal., 2002;Kommareddyetal., 2005;LeeandKim,2005). For systemic applications, the development of surface func- tionalizedandlong-circulatingNPsascellularprobesanddelivery agents is highly desired for passive targeting to tumors and inflammatory sites. PEG-modification of NPs affords long cir- culating property by evadingmacrophage-mediated uptake and removal fromthesystemiccirculation.Owingto its simplestruc- ture and chemical stability, it is a prototype of an inert and biocompatible polymer (Sperling and Parak, 2010; Verma and Stellacci, 2010). When bound to surfaces, PEG prevents other molecules tobindby steric effects. In fact, themolecules arenot attractedbyelectrostaticforcesandcannotpenetratethehydrated PEGlayer,producinganinerthydrophilicsurface.Moreover,PEG modifiednanoparticlesaremorestableathighsaltconcentrations and inbiological environments, avoidingnon-specificbinding to proteinsandcells (SperlingandParak,2010).This isparticularly important for invivoapplicationsbecauseoncetheNPsare inthe bloodstream, a portion of the plasma proteins that can adsorb to the surface (opsonins),may promoteNPs recognition by the mononuclearphagocyte system(MPS), andconsequently lead to rapidremovalof theNPs fromcirculation(BertrandandLeroux, 2012). Todate, there is a general consensus that to prolongNPs half-lifeintheorganism,PEGs’molecularweight,graftingdensity and chain architecturemust be optimized (Li andHuang, 2010; Grazúet al., 2012). For instance,Xie andcoworkers showed that MNPs functionalized with PEGwithmolecular weights higher than 3000Dawere not taken up bymacrophages in vitro,while extensive uptake was observed for PEG 600-coatedMNPs (Xie etal., 2007). Consequently, functionalizationofNPswithahighdensityof PEGofanadequate lengthnotonly increases thecolloidal stabil- ity of themodifiedNPsbut also their plasmahalf-life.However, toprovidePEGylatedNPswithtargetingandtherapeuticactivity, as well as with the ability of crossing different biologicalmem- branes, they must be conjugated with a variety of biologically relevant ligands, such as cell/tumorpenetrating peptides, tumor markers, and therapeutic agents (siRNAs, drugs). Concerning gold NPs, one of the main strategies is to assemble PEG and mixed biomolecule/PEGmonolayers on the nanoparticles’ sur- face. Liu et al. showed an escalation in the NPs’ stability with increasingPEGlength,decreasingnanoparticlediameter, increas- ing PEGmole fraction andmixedmonolayers prepared via the sequential addition of PEG followed by a peptide. In thisman- ner,NPsweremore stable than thoseprepared via simultaneous co-adsorption.ThesemodifiedNPswere able to target the cyto- plasm of HeLa cells, being the cellular uptake quantified using inductively coupled plasma optical emission spectrometry (Liu etal.,2007).Sanzetal.alsoobtainedpolyvalentPEGylatedAuNPs with a similar strategy. The authors developed an approach to attach specific biomolecules to the AuNPs’ surface and their effect in the functionalizationwithother specificderivatives.The effect of biofunctional spacers, such as thiolated PEG chains and a positive peptide (TAT) in dsRNA loading onAuNPs was reported. The authors hypothesized that the loadingof oligonu- cleotides onto theAuNP surfacemaybe controlled by ionic and weak interactions positioning the entry of the oligonucleotide through the PEG layer, by a synergistic effect of the TAT pep- tide andPEG chainswith specific functional groups, enhancing the dsRNA loading onto AuNPs (see Figure2) (Sanz et al., 2012). AnotherapproachtolinkbiomoleculestoPEGylatedAuNPsis making use of PEG as a spacer. This requires the use of bifunc- tional PEG chains that contain thiol at one end and a suitable functionalmoiety at the other (e.g. amino, carboxylate groups). Recently, Oh et al. described a different approach, where in a one-phase synthesis AuNPs were conjugated with PEG ligands yielding a narrow size distribution of highly stable NPs in the presence of high salt concentrations over a wide range of pHs (Oh et al., 2010b). One way or another, functional moieties of PEG ligands allow for further coupling of target biomolecules. Consequently, surfacemodificationofgoldclusters throughPEG spacers (Kanaras et al., 2002; Simpson et al., 2011)would allow themodifiednanoparticles to remain in the systemic circulation for prolonged periods and provide flexibility for efficient inter- action with a target. Besides, using a combination of different bifunctionalPEGspacers,goldnano-platformscanbemultifunc- tionalizedwith a variety of biologically-relevant ligands such as cell penetrating peptides, fluorescent dyes, tumor markers and siRNA(Condeetal., 2012a). PEGylatedQDshavealsobeensuccessfullyproducedforeffec- tive in vitro and in vivo circulation (Skaff andEmrick, 2003;Hu etal.,2010;Prowetal.,2012;Yangetal.,2012b).Recently,Poulose etal.developedhighlybiocompatiblePEGfunctionalized incad- miumchalcogenide luminescentQDs (CdS,CdSe, andCdTe) as animagingtool forearlydiagnosisofcancerbytargetingacancer cell line(Pouloseetal., 2012). AlthoughPEG is really useful toprolongNPs’ bloodhalf-life, it is known that in some cases PEG can hamper cargo release or hide other functional domains once the NP accumulates at thedesired target area (SawantandTorchilin,2012). Inclusionof Frontiers inChemistry | ChemicalEngineering July2014 |Volume2 |Article48 | 11