Seite - 21 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Condeetal. Biofunctionalizationandsurfacechemistryof inorganicnanoparticles affinity recognition, andcovalent coupling, eachofwhichhas its ownadvantages anddisadvantages. In this sectionof the review, the several coupling strategies for biofunctionalization of gold, MNPsandQDswillbeexamined. Covalentstrategies Covalent coupling provides stable and strong binding of the biomolecules to the NPs. Most proteins have amine groups in their surface, so they canbedirectly conjugated toNPs contain- ing reactive groups such as aldehydes, epoxides or anhydrides (Fuentesetal., 2005;Luetal., 2008). Ontheotherhand,couplingofNPsthatexhibitaminegroups with molecules containing aldehydes or epoxides can also be used.However, some biomolecules such as antibodies, oligonu- cleotides, carbohydrates or peptides do not include these func- tional groups, and should bemodified prior to the conjugation (Nobs et al., 2004). For instance, carbohydrates present in some antibodies can be oxidized using periodate to generate aldehyde groups that can reactwith the aminogroupspresent on theNPs surface (Fuentes et al., 2005). Nevertheless, chemical modifica- tionmay compromise biomolecules’ activity, so oneof themost frequentways to conjugatemolecules to theNPs is using linker molecules. EDC coupling reaction. 1-Ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC) is a zero-length crosslinking agent used to couple carboxyl or phosphate groups to primary amines, which may react with a carboxyl group of a biomolecule, forming an amine-reactive O-acylisourea intermediate. Addition of sulfo- NHS stabilizes the amine-reactive intermediate by converting it to an amine-reactive sulfo-NHS ester. The O-acylisourea inter- mediatemay also reactwith an amineona secondbiomolecule, producingaconjugateof the twobiomolecules joinedbya stable amide bond. This crosslinker has been used in diverse appli- cations, such as conjugation of carboxyl to amine groups in peptides and proteins, forming amide bonds in peptide synthe- sis, attachinghaptens to carrierproteins and form immunogens, labeling nucleic acids through 5′ phosphate groups and creat- ing amine-reactive NHS-esters of biomolecules (Grabarek and Gergely,1990). One of themain advantages of EDC coupling is water solu- bility, which allows direct bioconjugationwithout prior organic solvent dissolution. On top of that, the excess of reagents and by-products can be easily removed by dialysis or gel-filtration (Sheehan et al., 1965). However, the coupling reaction has to be carried out fast, as the reactive ester that is formed can be rapidly hydrolyzed in aqueous solutions. To increase the sta- bility of this active ester, N-hydroxysuccinimide (NHS) or N- hydroxysulfoxuccinimide (sulfo-NHS) can be used (Jang and Keng, 2008). Key parameters that should be controlled when usingEDCarepH(ashydrolysis is largelydependentonpH), the amountofEDCso thatNPsdonotaggregatedue to lossof elec- trostatic repulsive forces betweenNPs, and the ratio EDC/NHS (NakajimaandIkada,1995;Sametal., 2009;Shenetal., 2009). Using this protocol almost all kinds of molecules (i.e., enzymes, antibodies, peptides,DNA, fluorophores, etc.)may be attached to the nanoparticle surfacewithout priormodification FIGURE8 |EDCcouplingreaction.The1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC) is a zero-lengthcrosslinkingagentused tocouple carboxyl groups toprimaryamines. In thepresenceof N-hydroxysulfosuccinimide (Sulfo-NHS),EDCcanbeused toconvert carboxyl groups toamine-reactiveSulfo-NHSesters. Theadditionof Sulfo-NHSstabilizes theamine-reactive intermediatebyconverting it toan amine-reactiveSulfo-NHSester, thus increasing theefficiencyof EDC-mediatedcoupling reactions.Excess reagentandcrosslinkingby productsareeasily removedbywashingwithwater.OnceEDC iswater soluble, thecrosslinkingcanbedoneunderphysiologic conditionswithout addingorganicsolvent. (seeFigure8) (Pandey et al., 2007; Susumuet al., 2007; Rostro- Kohanloo et al., 2009; Conde et al., 2012a; Lavilla et al., 2012). For instance,usingtheEDCchemistry,Weisslederandcoworkers createdalibraryofMNPsdecoratedwithdifferentsyntheticsmall molecules for the development ofmagnetofluorescent reporters (Weissleder et al., 2005). Using these fluorescent MNPs it was possible to screen against different cell types or among differ- ent physiological states of a cell line. On the other hand, Sanz et al. have reported the effect of biofunctional spacers, such as thiolated PEG chains on the loading of RNAmolecules and a positive peptide functionalized by EDC coupling reactions on the surface ofAuNPs (Sanz et al., 2012). Lin and coworkers also used EDC to attach CH3O-PEG-NH2 to different types of car- boxylated NPs (MNPs, QDs) demonstrating that adjusting the ratio EDC/NP it was possible to prepare NPs with 0, 1, or 2 attached PEGmolecules (Lin et al., 2008). Similarly, Parak and co-workers alsousedEDC to attachNH2–PEG–NH2molecules, varying the molecular weight of the polymer on the surface of AuNPs (Sperling et al., 2006; Pellegrino et al., 2007). The covalent attaching of biofunctional short PEGmolecules to the polymer shell produces very stable particles in electrolytic solu- tion. This approach results in stablewater-soluble AuNPs (Sanz etal.,2012)andQDs(Ballouetal.,2004)withfunctionalgroups, e.g. −COOH or −NH2 on the free ends of PEG molecules. By controlling the EDC ratio, aggregationwas prevented. Dhar et al. have exploited the rapid intracellular uptake of AuNPs to deliverandactivatecisplatinandachieveefficientcytosolicdeliv- ery of platinum(IV) prodrug to lung cancer cells. The AuNPs used in this study were functionalized with thiolated oligonu- cleotides containing a terminal dodecyl amine for conjugation Frontiers inChemistry | ChemicalEngineering July2014 |Volume2 |Article48 | 21