Seite - 64 - in Cancer Nanotheranostics - What Have We Learnd So Far?

MorenoandPêgo AONcancer therapeutics aromaticbasesexposed(notburied insideadoublehelix),which confers a slight hydrophobic character to the molecule. These properties, enable some level of interaction with the cell mem- brane, which can be further potentiated by PS modifications, making possible, although still extremely inefficient, their use without the help of any vector formulations (Watts andCorey, 2012). This has been recently emphasized by the demonstra- tion that short LNAmodified AONs were able to sustain gene downregulation in a large variety of cell lines when adminis- tered in vitro unassisted by transfection agents (also referred as gymnotic delivery), although some cell lines still seem to be completely refractory to this type of AON uptake (Stein et al., 2010). The results obtained by gymnotic delivery seem to cor- relate well with the obtained in vivo gene silencing efficiencies for the “naked” LNAadministration; in fact, a better prediction of in vivopotencywas obtained in comparison to data resulting from transfection-mediated in vitroAONdelivery, amore stan- dardmethodtopreliminarilyanalyzeAONefficiency(Steinetal., 2010).Asimilarstudyshoweddownregulationofdifferentcancer genetargets,bythegymnoticdeliveryofLNA-AONinover30cell lines, althoughdiscrepanciesbetweenbothstudiesare seenwhen relating intracellular localization of theAONs (nuclear vs. cyto- plasmatic) and efficient down-regulation activity (Zhang et al., 2011). Despite several studies demonstrating some activity when using“naked”AONs invivo, andtheirwide tissuedistribution, it has alsobeen realized that these preferentially accumulate in the liverandkidneyandtoa lesserextent inspleen, lymphnodesand bonemarrow (Agrawal et al., 1995; Iversen et al., 1995;Graham et al., 1998; Geary, 2009; Straarup et al., 2010). Liver, as a pri- mary location of oligonucleotide accumulation has received a greater level of attentionwith someof themost promisingAON trials taking advantage of this effect, as seen withMipomersen (Hovinghetal., 2013).Liveraccumulationhasbeenattributedto the role of this organ in clearanceby the reticulumendothelium system(RES).This results fromtheabundantpresenceofphago- cytic Kupffer cells, together with the high blood flow received and, importantly, the existence of a fenestrated vasculaturewith anaverage100–200nmporediameterbetweenendothelial lining cells (Wisse et al., 2008). It should be noted that the pharma- cokinetics of AONs are dependent on chemistry, with themost favorable properties relating to the presence of PS linkages and the polyanionic character of themolecules. Thus, AONs based on PNA andPMOwhen administered as “naked” formulations invivo, arerapidlyclearedfromcirculationwhileshowingpoorer tissuedistribution(DirinandWinkler,2013). Tumor tissue also shares some of the abovementioned fea- tures, specially regarding its specificmicrovasculature character- istics (viz. for solid tumors). Fenestrations of 100–700nmhave been found in some tumor vessels, which together with a poor lymphatic drainage give rise to the enhanced permeability and retentioneffect(EPR)(Jangetal.,2003),responsiblefortheaccu- mulationofmacromoleculesornanoparticles intumors.Another effect to consider is the usually high interstitial fluid pressure (IFP) in tumors that obviates the normal rapid convective flow frombloodto the tissue interstitium(due toosmoticandhydro- static pressure differences). This effect is counterproductive in terms of drug accessibility to the tumor tissue, which then has to rely in slowdiffusionprocesses. Adense structure of intersti- tialmatrix and cells alsomounts a final barrier to the diffusion process (Chauhan et al., 2011). Finally, the uneven leakiness of vessels found in tumors further contributes toahighlyheteroge- neousprocessofdrugpenetration.Another consideration is that the larger thetumorthebigger theregionaldifferenceswithinthe tumor itself. This is illustratedby thepresenceof anecrotic core with an almost complete absence of blood flow, a seminecrotic regionwithpoorbloodflowwithinun-branchedvessels, a stable regionwithbranchedvessels andgoodflowandanactive angio- genic frontwherebloodflow is variable andcanbe substantially higher than insurroundinghostnormal tissues (Jain,2012). These hindrances can result inAONsdespite reaching tumor tissue, not being able to accumulate to a significant extent in the tumor tissue, with the additional drawback of distributing unevenly throughout the tissue (Plenat et al., 1995;Delonget al., 1997;Devietal., 2005). Certainly thesedelivery issues hamper amore effective trans- lationofanti-cancerantisenseoligonucleotides to theclinic. PERSPECTIVESONAONVECTORIZATIONFORCANCER THERAPEUTICS Given thewide tissue distributionproperties ofAONs and their preferential accumulation inorgansother thantumor tissue, this can leadto thenecessityofusinghighamountsofAONsinorder to reach ameaningful biological effect, raising concerns due to presenceofhighAONconcentrations inunspecifictissue/organs. Inaddition,althoughsomelevelof localizationto tumortissue is attained due to the EPR effect, there can be a large heterogene- ity in the targeting and distribution of AONs between tumors andwithin the same tumor.Not achieving a homogeneous and abundant distribution of AONs to the entire tumor can result in differential intracellular concentrations of the AONaffecting functional efficiencyandultimately leading tosomecells evading theanti-canceraction. Thedevelopmentofnanocarriers forAONdeliverycouldhave apositivecontributioninAONanti-cancerefficiencywhilemini- mizingtoxicity,althoughtheirutilitymustbeevaluatedinacase- by-case basis.Nanoparticle systemswill be also affected by inter and intra-tumorheterogeneity,wheredifferencesbetweentumor mass strongly influence theEPRandIFPeffects. In fact, theEPR effect is more prevalent in tumors of 100mm3 which limits its usewhentargetingsmallorunvascularizedprimaryorsecondary tumor (metastases) (Adiseshaiahet al., 2010).WhileAONsasso- ciated with nanoparticle systems can take greater advantage of the EPR effect, when “naked” administration is employed these will be affected to awider extent by IFP similarly to small drugs Interestingly, this could mean that free AONs could have an advantagewhendealingwithatumorwithalessdisturbedvascu- lar architecture orwhen tumor vasculature normalizationdrugs areused(Julianoetal.,2009;Chauhanetal.,2012).Thisviewcan, however,be toosimplisticas showninaworkdealingwith imag- ing andmodulation of AONmicrodistribution in solid tumor xenografts (Mocanuetal., 2007). Itwas seenthatadrug-induced decrease in IFPwas not accompanied by an expected improved distribution of the AON, in contrast towhat has been reported Frontiers inChemistry | ChemicalEngineering October2014 |Volume2 |Article87 | 64