Seite - 81 - in Cancer Nanotheranostics - What Have We Learnd So Far?

Conniotet al. Nanocarriers for immunecell targetingand tracking FIGURE5 |Thestealtheffect fromNPfunctionalizationwithPEG. (A) Particulate foreignentities inbodyfluidsarepromptly coveredwith opsonins, suchas the immunoglobulins IgGand IgAand thecomplement proteinsC3bC4b, inaprocesscalledopsonization.Opsoninsmark the particulateentity tophagocytosis through their recognitionbyFc receptors onphagocytic cells, suchasmacrophages. (B)FunctionalizationofNPs withPEGbygrafting, conjugationoradsorption—note the “mushroom-like” (a) or“brush-like” (b) configurationofPEG chains—providessteric stabilizationandstealthproperties, preventing the adsorptionofopsoninsat thesurfaceofnanoparticles.PEGhydrophilicity attractswatermolecules toparticlesurfaceavoiding theadsorptionof opsoninsatNPsurface, rendering them“invisible” tophagocytic cells. priortonanocarrierproductionmaybeadvantageous,sothatthe conjugation yield of the ligand to the polymer can be assessed and controlled.Nanocarriers can be thus producedwith awell- characterized (co)polymer and the density of ligands on their surface can be tailored. Physicochemical properties of the poly- mers must be evaluated after ligand conjugation, because the hydrophilic/hydrophobic balancemay be altered, particularly if macromolecules are linked(Betancourt et al., 2009; Sperlingand Parak,2010;Nicolasetal., 2013). The strategy of attaching ligandmolecules after nanocarrier production is usually applied, when antibodies, proteins and polypeptidesarechosenas targetingagents.As someorganic sol- vents are generally used in the preparation of nanosystems, this methodispreferredtoavoiddenaturationofthesecondarystruc- tureof the ligands.Also, since theyarebulkymolecules, theywill disturb the hydrophilic/hydrophobic balancewhich candifficult themethodofnanocarrierproduction(Nicolas et al., 2013).The drawbacks of this approach are related with subsequent purifi- cationof the formulationand its characterization.Theprocesses frequentlyused forpurification, suchas centrifugation,filtration anddialysis,maydegradeoralter thenanosystems.Additionally, it is usually difficult to prove that the ligand is covalently linked to the surfaceof thenanocarrier andnotonly adsorbed (Nicolas etal., 2013). Ligation strategies for functionalization. Several pathways have been developed to attach ligands onto nanosystems surface, suchas thecarbodiimide strategy, theMichael additionpathway, the biotin–streptavidin approach and theCopper-catalized liga- tionmethod (Betancourt et al., 2009; Nicolas et al., 2013). The native terminal groups of some polymers or specific moieties, introducedthroughchemicalmodifications,aregenerallyusedto applytheseschemesof functionalization.For instance,carboxylic acid terminals in aliphatic polyesters and poly(ethylene glycol) (Betancourtetal., 2009). Themost used scheme is based on the carbodiimide chem- istry. It relies on the coupling of a molecule containing a ter- minal aminegroupwithanotherwith anN-hydroxysuccinimide (NHS) ester end or an end group that can be easily esterified to NHSmoiety (Betancourt et al., 2009; Nicolas et al., 2013). TheMichael addition pathway is based on the thiol-maleimide coupling. Maleimide-polymers are used to produce nanocarri- ers, which are then decorated with thiol-containing targeting agents (Betancourt et al., 2009; Nicolas et al., 2013). However, the presence of native thiol groups in somemolecules, as pro- teins and peptides, is usually low (or absent in some cases) and many are hard to access. To overcome this, disulfide bonds can be reduced in thiol groups or heterobifunctional cross-linking agentsmaybeused(Nicolasetal.,2013).Thebiotin–streptavidin approach utilizes a strong non-covalent biological interaction betweenbiotinandavidin (Betancourt et al., 2009;Nicolas et al., 2013). Still, for this strategy, a targeting agent is usually chem- ically bound to avidin, which is a bulky glycoprotein that may thenobstruct the interaction ligand-receptor, essential for target- ing (Betancourt et al., 2009). TheCopper-catalized ligation is a highly efficientmethod, based on a cycloaddition reaction that fits in the“clickchemistry”classof reactions.Thechemical reac- tion is developed in mild conditions and with little or absent byproducts.Themajordisadvantageof thisapproach is theelim- ination of the Cu-based catalyst used for the reaction (Nicolas etal., 2013). Functionalization of nanosystems for immune cell targeting. Extensiveresearchhasbeenmaderegardingcell surfacereceptors in immune cells, the so-called PRRs. PRRs recognize pathogen- associated molecular patterns (PAMPS) and are involved in several stages of the immune response, from its initiation and proliferation, to its execution (Kumar et al., 2009). Different types of molecules may act as PAMPs, known as “danger sig- nals,” for instance lipids, lipoproteins, proteins, carbohydrates and nucleic acids. The recognition of PAMPs by PRRs triggers immune responses by activating multifactorial signaling path- ways. This leads to the induction of inflammatory responses mediated by several cytokines and chemokines (Kumar et al., 2009). Several classes of PRRs have been reviewed, such as TLRs, retinoic-acid inducible gene (RIG)-like receptors (RLRs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), DNA receptors (cytosolic sensors for DNA), scavenger receptors, and C-type lectin receptors (CLRs) (Kumar et al., 2009; Carrillo-Conde et al., 2011; Shen et al., 2013; Silva et al., 2013). Inmammals, themost studiedPRRclass is theTLRsclass. TLRsarepredominantlyexpressedbyAPCs,asDCs,but theyare also found on cells of the adaptive immune system, such as in αβT cells, regulatory T cells, and γδT cells, as well asNKT cells (Wesch et al., 2011). Through TLR activation, both the innate and the adaptive immune responses can be engaged, either by directactivationofTLRswiththeir ligandsonTandBcells,orby Frontiers inChemistry | ChemicalEngineering November2014 |Volume2 |Article105 | 81