Mucus models to evaluate nanomedicines for diffusion

Mucus models to evaluate nanomedicines for diffusion

Anne-Claire Groo

and Frederic Lagarce

1LUNAMUniversite´,INSERMU1066MINT(Microetnanome´decinesbiomime´tiques),Angers,France

2Ethypharm,Grand-Quevilly,France

3PharmacyDepartment,AngersUniversityHospital,Angers,France

In the fast-growing field of nanomedicine, mucus is often the first barrier encountered by drug products in the body, and can be the only barrier if it is not overcome by the drug delivery system. Thus, there is a need to design new nanomedicines that are able to diffuse easily across mucus to reach their pharmacological targets. In this design process, mucus diffusion studies are mandatory and have an important role in the selection of the best drug candidates. However, there is currently no standard procedure for diffusion studies across mucus. In this Foundation Review, we discuss the differences observed within mucus models and experimental protocols in diffusion studies, with an emphasis on nanomedicine diffusion.

Introduction

Colloids arebeingincreasinglydeveloped and usedto enhance the efficacy, and reduce the toxicity,ofdrugs.Inthispromisingareaofso-called‘nanomedicine’,somenewdrugformula- tionshavealreadyreachedthemarket[1]andthereisasubstantialamountofresearchunderway intonewcolloidalformulations toenhance their useasdrug deliverysystems. Forexample, encapsulationinnanodevicessuchasliposomesornanocapsulescanhelpthedrugtohavethe desireddistributioninthebody,thusenablingittoreachitspharmacologicaltargetinsufficient concentrationandavoidingothertissueswhereitcanbetoxic.Encapsulationalsohelpsthedrug toovercomebiologicalbarriers,suchastheintestinalepitheliumortheblood–brainbarrier.The journeyofacolloidalcarrierinthebodyiscomplexandhasbeenreviewedrecently[2];however, itoftenstartswithanencounterwithmucus.Infact,mucusisacomplexbiologicalmaterialthat lubricatesand protects manytissues. Giventhatmucus isubiquitous, colloidsystemsarein contactwithitinmanyareasofthebody,includinglungs,gastrointestinaltract,vagina,eyesand nasaltract.Thus,itisnecessarytocharacterizenanoparticlebehaviorinmucusduringtheprocess offormulationdesignandoptimization.

Irrespective of itsorigin, mucus comprises water(approximately 95%), glycoproteins (i.e.

mucins), lipids (0.5–5%), mineral salts(0.51%) and free proteins (1%) [3]; however, mucus displaysdifferent properties and finecompositiondepending on itslocationin the body. It inhibitspenetration bynumerousviruses [4]and isa usefulbarrier againstotherpathogens.

However,mucusalsoconstitutesapotentiallyefficientbarriertothedeliveryofnano-sizeddrug

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Anne-ClaireGroostudied pharmacyattheUniversityof Reims,Francefollowedby theformulationofcolloidal systemsattheUniversityof ParisXI,France.Sherecently earnedherPhDin pharmaceuticalsciencesfrom theUniversityofAngers, France,specializinginthe

nanocarrieroptimizationfororaldelivery.Shehas beenafellowatEthypharmSAsince2010.Hermain researchinterestsarethedevelopmentandthe evaluationofanticancerdrugnanoparticles,for crossingthemucuslayerandforimprovingoral bioavailability.

FredericLagarcereceived hisPhDin2004and,since 2012,hasbeenaprofessorof pharmaceuticaltechnology andbiopharmaceuticsinthe UniversityofAngers,France.

Heisalsoahospitalphar- macist,andsohisresearch hasatranslationalfocus(from benchtobedside).Hismain

interestisincancertherapy,especiallybioavailability enhancementbyexploitingtheinteractionsbetween drugproducts(mainlynanosystems)andlivingtissues.

Thisfieldinvolvesnotonlybiologicalbarrier-crossing studies,butalso

stabilityassessmentsofactivemoieties.Findingnew answerstomedicalneedsusinginnovativedrug formulationsiswhatdriveshimtoworkeveryday.

Correspondingauthor:.Lagarce,F. ([email protected])

deliverysystems.Thus,itisofprimaryimportancetodesignnano- carriers thatare ableto crossmucus, therefore,mucusdiffusion studiesareneeded.Despitethisneed,thereiscurrentlynostandard protocolavailableformucusdiffusionstudies;here,wedetermine whetherthedifferentmodelsusedtostudynanocarrierdiffusionin mucusaresimilarenoughtoeachothertoprovideapproximately thesameresultsorifthereisaneedforstandardization.Wefirst evaluatedifferencesandsimilitudesofmucusmodelsdescribedin the literature.Wethengo ontocomparediffusionmodels and evaluate the impact of experimental conditions on diffusion.

Finally,wehighlighthowphysicochemicalpropertiesofnanopar- ticlesinfluencetheirdiffusionthroughmucus.

Mucus models

There are various models of mucus described in the literature (Table1), fromthesimplestexvivo modeltothe closestinvivo models,andfromsimplemucin,artificialmucus,tonaturalmucus from horse,pigor human.It is alsopossible to use pathologic mucusandmucusproducedbyspecializedcellsforinvitrotrans- cellularcrossingoruptakeexperiments.Exvivoorinvivomodels havealsobeendescribed.

Thesimplestmodelsincludeonlymucinsolutionsreconstituted with different solutes. Norris and Sinko prepared reconstituted

mucinby mixing mucinwith sodiumphosphateand a sodium carbonatebuffer,adjustedtopH6.5[7].Bycontrast,Dawsonetal.

prepared reconstituted artificial pig gastric mucus by mixing piggastricmucin(PGM)60mg/mL,dipalmitoylphosphatidylcho- line(DPPC),bovineserumalbumin(BSA)andHepesbuffer(pH7.4) [12].Bhatetal.preparedamodelofcysticfibrosismucus(CFM)by addingcalfthymusDNAandBSAtoreconstitutedpiggastricmucus solution [16],whereas Bhat prepared a reconstituted piggastric mucussolutionbymixing PGM (40mg/mL) and isotonicphos- phatebuffer(pH7.4)containingsodiumazide,followedbytwo roundsofcentrifugationandthendialysis[58].Larhedetal.pre- paredanartificialmucusmodelcomprisingpurifiedPGM(0.4%),a lipidmixture(3%),pigserumalbumin(3.1%),DNA(0.5%),Tween 80(0.75%)and10mMphosphatebuffer[13].

Thus,thesedifferentpreparationprotocolsresultedindifferent mucusmodelsofdifferingpHandwithdifferentphysicochemical properties.Infact,mucusmodelsprepared fromdilutedmucin usingdifferentmethodologiesdifferednotonlyfromeachother, butalsofromcrudemucusmodelsextractedfromanimals.

Advantagesandlimitsofthemodels

Crudemucusisthemostidealmodelbutithassomedisadvan- tages. First, it is difficult to access a source of mucus from an individual. Second, the composition and, thus, chemical and

TABLE1

Mucusmodelsfoundintheliterature

Mucusmodel Origin Ref

Mucin TypeII/piggastricpurifiedmucin [5–8]

TypeIII/piggastricunpurifiedmucin [9,10]

Semipurified [11]

Artificialmucus Reconstitutedpiggastricmucus(BSA+DPPC+buffer) [12]

MLPD [13]

Reconstitutedpiggastricmucus(mucintypeII+lecithin+BSA+HEPES buffer,etc.)

[9]

Naturalmucus Ratintestinalmucus [14]

Piggastricmucus [15,16]

Pigintestinalmucus(PIM) [5,8,13]

Horserespiratorymucus [17]

Humancervico-vaginalmucus(CVM) [18–26]

Humanairwaymucus(HAM) [27]

Naturalbutpathologicmucus Cysticfibrosissputum(CFS)orcysticfibrosismucus(CFM) [16,23,28,29]

Chronicrhinosinusitismucus(CRSM) [30]

Invitromodel HT29-MTX [31–37]

HT29-FU [34]

HT29GlucH [38]

HT29-H [39,40]

Calu-3 [41]

Co-culture Caco-2/HT29-MTX

(relative%ofeachcelllinegiveninthenextcolumn)

90/10 [37]

75/25 [37]

76/24 [35]

70/30 [42]

50/50 [37,43]

Co-cultureCaco-2/HT29-H 50/50 [44]

Tissuesexvivo Porcinenasalmucosa [45]

Ratjejunumportion [46–48]

Ratintestine [49]

Ratileum [50,51]

Invivomodel Rat [32,46,47,50,52,53]

Mice [54–57]

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physicalpropertiescanvarybetweenbatchesbecauseofinterin- dividual variability; for example, mucin concentrations varied from102to473mg/mLamongsixdifferentmucussamples [29]. To limit interindividual variation, some batches can be collected and mixed. Mucus samples can be stored at 208C without significant change. For example, no effect of freezing andthawingonviscoelasticity[29]wasobservedandstorageat 208C did notinfluence the diffusioncoefficients ofdrugs [5].

However, a little evaporative loss from cervicovaginal mucus (CVM)wasobservedatroomtemperatureandstorageat208C reducedevaporativeloss[59].

Pigisa relativelylargeanimaland soitispossible toobtain sufficientmucusfromonlyfewanimalstoperformseveralexperi- ments. Moreover,pig mucus and human mucus are similar in structureandmolecularweight[60],whichisimportantgiventhat itisalsopossibletoobservedifferencesin mucuspropertiesfor differentanimalsofthesamespecies[61].Asaresult,commercial pigmucinhasbeenusedasamucussubstitutetopreparemucin solutions.TwoformsofPGMarecurrentlyavailablecommercially:

purifiedmucin(typeII)andunpurifiedmucin(typeIII).Themain advantageisthatthecompositionismorestable,althoughthisis not always relevant given that mucus contains various other components,suchaslipids,proteins,orsalts.Thus,someresearch- ershave focusedonreconstitutedmucus;forexample,Dawson etal.[12]mixedPGMwithBSA,DPPCandbuffer,whereasMcGill andSmyth[9]mixedmucinwithlecithin,BSAandHEPESbuffer;

an artificial mucus model was also proposed by Larhed [13].

Interestingly,reconstitution ofmucuswith mucindid notsup- pressitsvariabilitycomparedwithcrudemucus,inthatMcGill andSmythobservedsomeheterogeneityincompositionandnon- uniformity of the rehydrated mucin polymers used in in vitro preparedmucusmodels[9].

Griffithsetal.demonstratedthattheextractionprocessmodi- fies commercialPGM samples. It disruptsthe disulfide bridges, leadingtoaweakersol–geltransitionataroundpH4andalackof gel formation [10]. The structural perturbation in mucin was confirmedbythelackofinteractionsbetweenmucinandpoly(- ethylene glycol) (PEG). Bhat et al. compared drug diffusion throughCFMand throughgel and solfractionsgenerated bya separationprocess.Unfortunately,thisprocessalteredthemucin structure,asevidencedbythehighlybranchedstructuresobserved bytransmissionelectronmicroscopy(TEM)[16].

In vivo and ex vivo assays canbe consuming in terms ofthe numberof animals required.However, working with slaughter- housesenableslarge amountsofanimalmucosato beobtained fromonlyafewanimals.Forexample,Wadelletal.studieddiffusion throughporcinenasalmucosa[45].Thismucosawaslargeenough andonlyoneindividualwasusedtoachievetissuespecimensfora fullsix-chamberexperiment,whereasahighernumberofsmaller animalswere neededto getthesamearea.However,forstudies involvingintestinalmucosa,feweranimalsareneeded.

Modelcomparisons

Studies compared molecule diffusion through different mucus models and showed different apparent permeability according tothemucusmodel.Mucuscompositiondependsontheorigin of the mucus (species and organ source) and its composition influencesitspropertiesandreactivityagainstothersmolecules.

Theweight-averagemolecularmassofmucincomprisingmucus isdifferentdependingonthefunctionofitsoriginallocation[62].

For example, the molecular mass of pig colonic mucin is 5.510⁶Da, of human cervical mucin is 1110⁶Da and of PGMis4410⁶Da.Theinfluenceofmucusmodelcomposition onmucusproperties haslargelybeenstudied overthepastfew years,withobviousdifferencesreportedbetweenpathogenicand nonpathogenic models. For example, chronic rhinosinusitis mucus (CRSM) and cystic fibrosis sputum (CFS) have similar barrierpropertiesbecausetheviscoelasticityofmucusgelisexa- cerbated in both cases by pathogenic inflections and chronic inflammation. Asa consequence,results of Lai etal. suggested that CRSMhas greateradhesivitycomparedwith healthyCVM [30]. The solution environment also has an impact on mucus properties, particularly in the case of disease. For example, in CFM, the high extracellular Ca²⁺ concentration leads to thick mucusoverthelongterm[63].Thepoorbicarbonateavailability inthismucuscanexplainitshighviscosityandmucinaggrega- tion,becauseoftheabilityofbicarbonatetosequesterCa²⁺[64].

Thehighlevelsofsolubleproteinsonthemucosapartiallyexplain thecharacteristicallythickmucusinasthmaandotherbronchial inflammatorydiseases[65].

PurifiedPGM solution did notprovidean accuratemodelof nativemucusbecauseitdidnotexactlyreflectmucusconstituents suchaswater,mucinandlipids,mineralsaltsandfreeproteinsin either their quality or quantity, which increased the possible interactionsbetween particlesand mucus.Larhed etal. studied the diffusion of different drugs through native pig intestinal mucus (PIM) and purified pig gastric mucin (PPGM) [5]. They demonstrated that the diffusion coefficient of lipophilic drugs wasreducedinanativePIMmodelbutlesssoinPPGM,compared with the diffusion coefficient in buffer. For example, 36% of cyclosporine A diffused in PPGM and 16% in PIM. The same phenomenon was observed with another drug: 78% of 1-dea- mino-8-D-arginine-vasopressin (dDAVP) diffused in PPGM and only17% inPIM.Thus,a substantialpartofthe mucusbarrier waslikelytobeformedbyothercomponentsofthenativemucus besides mucin. Moreover, a relation between lipophilicity (i.e.

logP) and diffusions in PIM wasobserved but norelation was foundinPPGM.Thus,thenativePIMwasamorerealisticmodelof gastrointestinalmucusandprovidedmoreinformationregarding the barrier properties of mucus in vivo. Similarly, McGill and Smyth’sstudyshowedthatrhodamineBpermeationwassignifi- cantlydifferentinmucinsolutionandinanartificialCFSmodel becauseofthedifferencesinthecompositionofthemucusmodel [9].

Components other than mucin have also been found to be responsibleforthe reduceddiffusionoflipophilicdrugs inPIM comparedwithPPGM.Larhedetal.identifiedlipidsandproteins ascomponentswithanimportantimpactondrugdiffusion[13], withthedrugsinteractingwiththelipidsandproteins.Thesame authors triedto reproduce artificialmucus with a composition mimickingPIM.However,thediffusionobtainedintheirartificial mucuswassimilarbutnotidenticaltothatinPIM,highlighting thefactthatitisdifficulttoreconstitutethecompletestructureof nativemucus.

Bhatetal.comparedthediffusionofthreedrugsthroughbuffer, native mucus and synthetic mucus models[16]. For the drugs

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tested,theobservedpermeabilitywasalwaysinthefollowingrank order: buffer>reconstitutedpiggastricmucus(i.e. mucinsolu- tion)>whole CFM>CFM sol fraction>synthetic CFM>CFM gel fraction. Synthetic CFM solutions wereprepared by adding BSAandDNAtopiggastricmucussolutions.Lielegetal.observed thatmucinconcentrationwasanessentialparameterfordiffusion:

ifmucinconcentrationincreased,theimpactofthebarrierbecame morepronounced[66].

Mucinconcentrationcanvarywiththelocationofmucusinthe body,aswellaswithvariousotherphysiologicalandpathological parameters[67].Griffithsetal.studiedthediffusionofpolymersin mucinsolutionsthatrangedinconcentrationfrom0to5%(w/w).

Adecreaseinthediffusionratewasshownwithincreasingmucin concentration[10].Therefore,diffusiondependslargelyonmucus componentsanditsproportions,whichcanbeexplainedbythe factthatinterfiberspacingdependsonlyweaklyonhydrationbut moreontheconcentrationofmucin[21].

In addition to observed differences between mucus model compositions, differences in mucus model structure have also been observed and have a role in particle and drug diffusion.

TheaverageporesizeofhumanCVM,determinedbyfittingthe measured diffusion rates of particles to Amsden’s obstruction- scalingmodel,was34070nm[18].Theaveragemeshspacing of humanCFS was14050nm,asshown by the dynamicsof mucus-resistant particles [28]. However, comparisonis difficult becauseresultsdependonthesourceofmucusaswellasonthe methodofsamplepreparation,whichcan disruptmucusstruc- ture. Ensign et al. observed variations in the mucus mesh at differentanatomicallocations[68].

Thesamplingmethodusedisalsoimportant,giventhatmucus comprisestwolayers,thefirmlyadherentmucusandtheloosely adherentmucus[69].Iftheextractionmethodistooenergetic,the samplewillcontainremnantsofmucosa,suchascellsandDNA, whereas,ifthesampleistoosuperficial,itmightcontainonlythe freemucuslayer.

The increased hydration of ovulatory endocervical mucus (OCM)comparedwithothermucussecretions(duringnonovula- toryperiodsandatothermucosaltissues)increasedtheporesizeof mucin.Tangetal.performedstudiesofPLGAnanoparticlediffu- sioninOCMandCVM[23].DifferencesbetweenOCMandCVM ledtoadifferenceinthepenetrationimprovementresultingfrom the PEG coating. A modest increase in penetration rate was observedinOCM,whereasthesamemodificationimprovedthe penetrationrateinCVM400-fold.

As discussed above, mucin is often used to prepare mucin solutionsor artificialmucus,althoughthepreparationmethods usedtoobtainmucincandisruptitsstructure.Therefore,evenif mucinisusedatthesameconcentrationasinnaturalmucusand withtheothercomponentsofmucus,differencescanbeobserved comparedwithcrudemucus.Forinstance,anionicparticlemobi- litywassignificantlyhigherinpurifiedPGMthaninnativeintest- inalmucus[8].Thedifferenceinmeshstructureofnativemucus comparedwiththatofpurifiedmucinand/ordifferencesincom- positionbetweenthetwomediawererelatedtothisdifferencein mobility.Thedegradationoccurringduringthepurificationpro- cedurehasbeenrelatedtooneorotherofthesedifferences.Mucin waspresentathighconcentrationsinbothmedia,and cationic particlemobilitywassimilar,owingtotheadhesionofthecationic

particles to negatively charged mucin fibers. Particle transport ratesweremoreheterogeneousinnativemucus,becauseofthe higherheterogeneityoftheporosityofthemucusmesh.Themore homogeneousnatureofthepurifiedmucinsolutionversusnative mucuswassupportedbymicroscopicobservation.

Nanoparticletransportbehaviorwasnotsignificantlydifferent incolonicmucusonthesurfaceoffreshlyexcisedmousecolon tissuescomparedwithmucusscrapedfromthetissuesurface.This suggests that the collected mucus layer was, in this case, not disturbed[68],and enabledthe researchers tostudy the mucus barriereffectonly.

Diffusion systems

Variousprotocols havebeen developedtoevaluate interactions betweenparticlesandmucus(Table2),suchasthemucoadhesion assay[48,70,71],invivoexperimentations[32,55]withradioactiv- itystudies[53]orpharmacokinetic(PK)studies[46,54,72],binding proprieties[73]anddiffusionstudies.Here,wefocusondiffusion protocolsand brieflydiscussmucoadhesion, whichis oftenthe firststepofdiffusion.

Tostudydrugand particlediffusion,numeroussystemshave beenused,including multipleparticle tracking(MPT)[8,12,18–

20,22,23,25,28,74–76];two samples tubes that arethen filtered and centrifuged [11]; side-by-side systems [6,16]; side-on-three compartment diffusion [15]; diffusion chambers [45]including Ussingchambers[46,47,49,77];modifiedFranzdiffusioncells[14];

modifiedTranswell-Snapwell¹diffusion chambers[7,29];modi- fieddiffusion cell setups [9]; fluorescencerecovery after photo- bleaching (FRAP) [21]; radioactivity with two syringes [5,13];

Transwell¹ covered by cells [24,41,78]; Transwell¹ diffusion [32,33,35–39,42,43,50];andcellassociation[79].

Side-on-threecompartmentdiffusionisoneofthemostcom- monly used methods. The diffusion cell comprises one donor compartment,oneacceptorcompartmentandonecentralcom- partment containing the mucus model. Drugs or particles are placedinthedonorcompartmentandtheirarrivalintheacceptor compartmentisevaluatedovertime.Giventhatmucusisplaced betweentwo compartments,theamountofdrugor particlesin thiscompartmentdeterminestheirpermeabilityordiffusioncoef- ficientthroughthemucus.

Membranesbetweencompartmentsareimpermeabletomucus butnottodrugsorparticles;thus,theircapacitytoretainthedrug orparticlesmustbewellknowntodistinguishtheeffectsofthe membraneversusthemucusdiffusion.Therefore,differentside- on-three compartments have been developed, in the form of diffusionchambers(i.e.side-by-side¹diffusioncells)customized withamembraneholder(Fig.1)[6,16].

Numerousresearchteamshavedevelopedin-housemanufac- tured side-on-three compartment diffusion cells. For example, Shaw et al. added a polycarbonate filter membrane and metal gauze filtersto a diffusion cell [15].Norris and Sinkomodified Transwell-Snapwell¹ chambers, comprising two compartments andatissuebetweenthem,byaddingfiltersandaringonwhich toplacemucus[7],asdidSandersetal.[29].Similarly,Grubeland Cavemodifieda microfiltrationdeviceto obtaina permeability device[11]. When studiesfocus on mucosal tissue, the experi- mental systems used are simpler because tissue is more easily

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maintained between compartments. For example, Bravo-Osuna etal.usedaUssingcellcomprisingtwocompartmentsseparatedby ratintestinalepithelium[77],whereasWadelletal.placedporcine nasalmucosaindiffusionchambers[45].

AnotherapproachwasusedbyLaietal.[19].Particletransport ratesweremeasuredbyanalyzingtrajectoriesoffluorescentparticles inmucusbulk,byMPT.Inthiscontext,onlyonecompartmentis needed, thus avoiding the membrane affecting diffusion. The microscopicmotionofhundredsoffluorescentparticlesisrecorded byvideomicroscopyand,thus,particledetectionisperformedin mucuswithoutdisturbingthesystem.Inthesameway,FRAP[21]

hasbeenusedtoinvestigatethemobilityoflabeledmoleculesin mucusandbiogels. Thesampleisplacedonamicroscopeanda high-intensitylaserbeamisusedtobleachthefluorescenceofthe molecules,whichcausesadropinthefluorescenceintensity.The diffusion coefficient is obtained by the recovery profile of the fluorescence intensity, which results following diffusion of the nonbleachedmoleculesintothisarea(Fig.2)[80].

As a noninvasive method, pulsed-gradient spin-echo NMR (PGSE-NMR) was used by Griffiths et al. [10] to quantify the diffusionofaprobepolymer.Thecentralfeatureofthistechnique isthe applicationofamagnetic fieldgradientthat encodesthe positionofthemoleculeintotheNMRsignal.

Otheroriginalmodelscanbeusedtopredictparticlediffusion through mucus. For example, a 2D model was developed for

studyingthe interactionof surface-modifiedlipid nanocapsules (LNC) with mucus. This 2D model, based on surface balance measurements at a constantpressure or area,can be usedas a screening method for choosing suitable surface-modified LNC formulationsforassessingdiffusionusing3Dmodels[81].

Advantagesand limitsofdiffusionsystems

Thelimitsofdiffusionprotocolsarerelatedtothequantificationor detectionmethodsused.Someteamshaveusedradiolabeleddrugs anddetectedresultingradioactivitybyusingaliquidscintillation counter.Forexample,Bravo-Osunaetal.used¹⁴Cmannitol[77]

and Wadelletal. used¹⁴C mannitol and D-(2-3H)glucose[45], whereas Larhed et al. used ¹⁴C mannitol, ³H propranolol, ¹⁴C hydrocortisone, ³H testosterone and other radiolabeled drugs [5,13].Bycontrast,radiolabeled¹⁴CibuprofenwasusedbyShaw et al. [15]. In addition, Saltzman etal. labeled molecules with fluorescein and measured their diffusion coefficients by using computer imaging of fluorescence profiles and by FRAP [21].

However,workingwithradioactivityisexpensiveandisnoteasily accessedbecauseoftheneedforspecificequipment;inaddition, agreementandsafetyrulesarebothnecessaryandstringent.

MuchequipmentisrequiredforMPT,includingasilicon-inten- sified target camera mounted on an inverted epifluorescence microscopeequippedwitha100oil-immersionobjectivelens;

the appropriate filters; glass chambers; specific software; and TABLE2

Evaluationofcolloiddiffusionwithdifferentmodelsrelatedtotheirinvivoefficacy(predictability)

Route Invitromodel Exvivomodel Invivomodel Ref Drugnanocarrier Predictability/conclusions

Oral Caco-2/HT29-M6 Efficiency [82] Encapsulationofcalcitonin

intochitosannanocapsules

Invitromodelrevealedthatthe mucoadhesivepropertiesofchitosan nanoparticlesmayrepresentakeyfactorfor theirabilitytoimprovepeptideabsorption afteroraladministration

Ussingchambers (ratjejunum)

Invivo bioadhesive studyand pharmacokinetic (PK)

[47] Paclitaxel(PTX)-loaded pegylatednanoparticles(NP)

Similarimprovementofbioavailabilitywas observedforPEGPTX-NPinvitroandinvivo

Mucinadhesion (mucintypeIII)

PKstudy [83] PTX-loadedchitosan–vitamin Esuccinate(CV)nanomicelles (chitosanthiolatedornot)

ThiolationimprovefourfoldAUCforCV nanomicellesandleadonlytoatwofold increaseinmucinadhesion

Muco-adhesion PKstudy [84] Enoxaparinloaded

nanocomplexes(chitosan graftedglycerylmonostearate copolymers)

Mucoadhesionresultsarenotshowed(only:

mucoadhesionsignificantlyincreasedwith modificationofchitosanwithGMcompared withthatofchitosan,GMgraftratio:

3.7%=11.1%>18.6%andchitosan 100kDa>20kDa)

Vivo:C<3.7%<11%>18%and 100kDa>50kDa

Sameconclusion:maximalbioavailabilityfor nanocomplexespreparedusingCS100- GM11.4%copolymers

Colon MPTinmucus Trackingonfreshly excisedmucosal tissues

[68] Displacementofparticlewerenot

significantlydifferentincollectedcolonic mucusandinexvivocolontissue

Pulmonary MPTinCFS Invivoairway

genetransfer

[85] DNAnanoparticlescomposed ofplasmidDNAcompacted withblockcopolymerofpoly-

L-lysineandPEG(2,5,and 10kDa)

AllDNAnanoparticleswereimmobilizedin freshlyCFS.

MicereceivingCK30PEG10korCK30PEG5kDNA nanoparticlesexhibitedhigherluciferase expressionthanCK30PEG2k(duetohigher nucleaseattackofCK30PEG2k)

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fluorescentparticles.Laietal.[18,19]andSuketal.[28]alsoused fluorescent polystyrene (PS) particles obtained from molecular probes.Usingthesamemethod,CraterandCarrierusedfluores- centFluoSpheres¹obtainedfromInvitrogenmolecularprobes[8].

Tangetal.[23]preparedfluorescentPLGAnanoparticles.Unfortu- nately, fluorescent labeling can modify carrier properties and, thus,theirdiffusionability.

Inadditiontoquantificationordetectionmethods,thediffu- sion system has a role in diffusion evaluation. Experimental variability was observed by Bhat et al. [16] using a modified side-by-sidediffusioncellandmucusplacedinanapproximately 3-mmthickchamber.Thisthicknessishigherthanisfoundinvivo.

Withasimilarsystem,NorrisandSinkoobservedthesamevaria- bility in the measurements with a smaller mucus thickness of 0.38mm[7].Thevariabilitywasnotonlyrelatedtothethickness ofmucus,butalsotothecomplexityofthediffusionsystem.In fact, given that the membrane is a supplementary barrier to diffusion,itschoiceisimportant.Thegoalshouldbetoselecta membranewiththelowesteffectondiffusiontoobservephenom- enapurelyrelatedtomucus.Intheirstudy,Bhatetal.showedthat drugdiffusionwasundermembranecontrolforthethreedrugs thattheytested[6].Inthecaseofdiffusionacrosscompartments, whenthedonorsolutionisplacedinthedonorcompartmentat thestartoftheexperiment,theemptymembranebecomessoaked bysolution,correspondingtothetimeofflowestablishment[86].

Steady-stateconditionsarethenestablished.Membranethickness should be the lowest and experimentation duration should be highenoughtooverlookthetimeofflowestablishment.Experi- mentationdurationshouldalsobelongenoughtoenablemem- brane equilibration,but also shortenough to avoidsignificant concentrationvariationsinthedonorsolution.Insuchasteady- statecondition,equationofpermeabilitycanbesimplified.Using theseparticularconditions,Bhatetal.determinedpermeationat steadystatefordrugdiffusionacrossCFM[16].

TheapparentpermeabilityPapp,expressedincms¹,isclassi- callycalculatedusingEq.(I)[7,14,45,51,77]:

P_app¼ dQ

AC₀dt (I)

wheredQ/dtistherate ofdrugappearanceonthereceivedside (mg s¹), C0 is the initial concentration over the donor side (mgmL¹)andAisthesurfacearea(cm²).

ItisimportanttonotethatEq.(I)isvalidandcanbeusedonlyif the membrane volume is negligible versus the volume of the compartments;thatis,l<0.02.However,ifeitherthelengthof membraneequilibrationormembranevolumeistoohigh,Eq.(I) shouldbemodifiedusingl[87].

Some researchers have used Eq. (II), which is based on this previous formula,but that takesintoaccount the contribution ofeach barrier[6,16].Incaseof multilayersystems, membrane permeability,P,isgivenbyEq.(II):

P¼D d 1

P_total¼X1 P¼Xd_i

D_i (II)

whereDisthediffusioncoefficientofthemolecule,expressedin cm²s¹.Thethicknessofthelayerdisexpressedincm.Aside-on- threecompartmentsystemcontainstwomembranesandamucus layer,theformerofwhichcanconstituteanimportantbarrierto diffusion[88].Inthiscasethepermeabilitycanbeexpressedas:

1 P_total¼ 1

P_mb1þ 1 P_mb2þ 1

P_mucus; (III)

wherePmb1andPmb2representthepermeabilityacrosseachofthe membranes,Pmucusisthepermeabilityacrossthemucuslayerand P_totalisthepermeabilityacrosstheentiresystem.

In the case of a lack of system uniformity, a concentration gradient appearsand constitutes a pseudo-membrane [89].The existenceof unstirred layersimplies that, in any phenomenon dependingonthedifferencebetweenthetwosurfaceconcentra- tions,possibleseriouserrorscanbemadebyusingthedifference betweenthebulkconcentrations.Theeffectoftheunstirredlayer

Fluorescence F(i)

F(∝)

τD

0 Time

t = ∝ t = t

t = 0 t < 0

F(0)

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FIGURE2

Schematicrepresentationofafluorescencerecoveryafterphotobleaching experiment.TheinitialfluorescencebeforebleachingisrecordedasF(i).At t=0,adropinfluorescencetoF(0)iscausedbyahigh-intensitylightbeam bleachingthemolecules.Thebleachedmoleculesexchangetheirpositionin thebleachedareawithnon-bleachedfluorescentmoleculesfromthe surrounding,duetotherandommotion/diffusion.Thisresultsinarecoveryof theobservedfluorescence.

Figurereproduced,withpermission,fromOcchipintiandGriffiths[80].

Excess solution

Receiver

Custom membrane holder

Filling syringe Donor

Star-shaped stirrer Circulating

water in (37°C) Circulating

water out (37°C)

Sampling port

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FIGURE1

Side-by-sidediffusioncellwithacustomizedmembraneholder.

Figurereproduced,withpermission,fromBhatetal.[6].

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ismorecomplicatedthanthecaseofstirredlayer,andisgivenby Eq.(IV):

1

P_total¼X 1 P_mbþ 1

P_mucusþX 1

P_conc; (IV)

where Pconc is the permeability across the pseudo-membrane togetherwiththeconcentrationgradient.Forexample,Korjamo etal.observedthat,byvaryingstirringspeed,theconcentration gradienthadadifferenteffectondrugpermeabilityacrossCaco-2 cellsonTranswell¹[90].

Themethodbecomesveryinaccuratewhendiffusionisalmost entirelyratecontrolledbytheunstirredlayers[91].Thisiswhy,if the concentration in each compartment varies substantially betweenthestartandendoftheexperiment,stirringisnecessary tohomogenizethemedia.

Parametersinfluencing diffusionand comparisons

Diffusioninmucusdependsonitscomposition[13],suchasthe mucinconcentration[66],which,asdiscussedabove,dependson themucusmodelused[8].Thus,thechoiceofmucusmodelis crucial.Themucusmodelusedneedstobetheclosesttothetype ofphysiologicalmucusencounteredbythedrugdeliverysystemin vivo.Particlediffusionalsodependsonthesurfacechemistryofthe particle[8]andtheparticlesize[22].However,otherparameters canalsoinfluencediffusionandshouldbechecked.

GrubelandCaveobservedthattheeffectoftheformulationon mucusviscosity appearedto determinethe movement of clari- thromycinthroughmucus[11].Forexample,thegreaterpropor- tionofinactive polymericingredientsin Biaxin¹granules,the higherthe increaseinthe viscosityandthe moreenhancedthe barrierpropertiesofgastricmucin.Moreover,Sandersetal.showed thatthe elasticmodulusofmucusinfluencedthepercentage of transportednanospheres[29].

When Griffiths et al. added dendrimers to mucin solutions, changes in the mucin scattering were induced, indicating an interaction betweenthese polymersand mucin [10],a change thatwaspHdependent.Asaconsequence,thediffusionofpoly- mers showed a complex dependency on both pH and mucin concentration.Caoetal.demonstratedthatmucusundergoesa pH-induced conformation change [92], whereas Lieleg et al.

showedthatincreasingthemucuspHfrom3to7resultedina generalincreaseinparticlemobilitybecauseacidicmucusformed ahigherandmoreselectivebarriercomparedwithneutralmucus [66].GiventhatmucuspHvarieswithitsfunctionand,therefore, localizationinthebody(Fig.3),thechoiceofmucussourcetoset upamodelhastobemadecarefully.

Shaw et al. demonstrated that the diffusion of ibuprofen increasedforhigherpHvalues,asaresultofchangingtheelectro- staticrepulsioninteractionandloweringtheviscosityofmucus [15].Interactionswererelatedtotheionizedstateofthemucus andofibuprofen.Nochangewasobservedforparacetamol,which isaunionizeddrug.Therefore,theeffectonthechargeinteraction betweenthedrugandmucusismoreimportantthantheeffectof viscosityondiffusioninthisstudy.Lielegetal.foundthatelec- trostaticinteractionsweresensitivetotheioncontentofasolu- tion. Given that the surface charges of synthetic particles or polymers were partially shielded by solubilized ions in buffer, thestrengthoftheattractiveorrepulsiveforcesbetweendiffusing particlesandmucusdependedonthesaltcontent[66].

Theconcentrationofthestudiedparticlesisalsoimportant.For example, Lai et al. demonstrated that the addition of a high concentrationofparticlestoCVMpreventedtheirtransportand causedthecollapseofthemucusfibers,whereasalowconcentra- tion of particles did not cause bundling and allowed particle movements[19].At highPSparticleconcentration(i.e.10%,v/

w),hydrophobicinteractionscancausetheaggregationofmucin fibersinhumancervicalmucus(HCM)[26].Similarly,Wangetal.

observedthattheeffectofmucoadhesivenanoparticlesonmucus dependedontheparticleconcentration[76].

Therefore,drugandparticlediffusionaresensitivetopH,ionic force,viscosity,particleconcentration,andtheexperimentalcon- ditionsofdiffusionstudies(i.e.whenandhowlongdiffusionwas observed). Given that experimental conditions can change between researchers and teams, comparisons between studies mustthereforebemadewithcaution.

The specific case of colloid diffusion through mucus

Knowingtherelationbetweencolloidalcarrierpropertiesandtheir ability to diffusein mucus enables betterdesign of these drug deliverysystems.Here,wediscusseachparameterthatshouldbe optimizedtogainbettercolloiddiffusioninmucus.

Colloid design:sizechoice

Moststudiesconcludethatsmallerparticlesmovefasterinmucus [52].Forexample,Sandersetal.observedadifferenceinthetrans- portofPSnanospheresthroughCFS,dependingonparticlesize.

Increasingparticlesizefrom124nmto270nmor560nmdecreased themeanpercentofnanospherestransportedafter150min,caused mainlyby stronger stericobstruction[29].Similarly,Norris and Sinkoobservedthelimitedabilityofparticles>0.5mmtodiffuse throughmucus[7].Bycontrast, forHosseinzadehetal., alarger

Acidic Alkaline

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FIGURE3

MucuspHrelatedtoitslocalizationinthebody.

Figureadapted,withpermissionfromLielegetal.[66].

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surface area was provided by smaller nanoparticle sizes, and increasedadsorptioninmucin,whichledtohighermucoadhesive propertiesformucoadhesivenanoparticles[70].Otherstudies[9,51]

confirmed the relation between size and mucoadhesion, which influencesparticlediffusion.

Sizeeffectmightbetheresultofastericobstructionofmucin meshspacingthatislinkedtomucininterfiberspacingsize.This size dependson themucus modeland the methodusedforits determination.Forexample,Saltzmanetal.determinedthegeo- metriccharacteristicofHCMgelsbyscanningelectronmicroscopy (SEM)anddiffusionstudies[21].Theprobableinterfiberspacing, assuming a random fiber arrangement, was 170nm and the inferredinterfiberspacingobtainedbymeasuringdiffusioncoeffi- cient was 150nm. Olmsted et al. predicted a mesh spacing of 100nm, by applying Amsdem’s obstruction-scaling model to HCMandbyelectronmicroscopy[26].Yudinetal.revealedthat mucus hasa fibrousstructurewitha 500-nm interfiberspacing betweentheprimaryelementsand anadditionalfinerstructure withaspacingofapproximately100nm[93].Similarly,Kirchetal.

observedbycryogenicSEMthathorserespiratorymucushadlarge poresheterogeneouslycombinedwithverysmallpores[17],which is in accordance with findings for CVM [18]. In SEM images, normalHAMporesrangedfromtenstohundredsofnanometers indiameter,withmanypores<100nm[27].

Colloiddesign:considerationsofsurfaceproperties

Aswe discuss below, surface properties suchaselectric charge, chemicalmoieties,hydrophilicityand/orlipophilicityhavebeen showedtohaveasignificantroleintheabilityofcolloidalcarriers formucusdiffusion.Thus,anegativerelationbetweenthediffu- sionofpeptidesandtheirlipophilicproperties(i.e.logP)inPIM hasbeenobserved[5].Mistryetal.alsodeterminedthatpolysor- bate-coated PS nanoparticles increased transport in mucus by increasinghydrophilicity[55].A surfacemodification ofthe PS particlewithpegylatedpolysorbate,increasedtransportthrough mucus not only by increasing the hydrophilicity, but also by reducing the negative charge of the PS particle. Wang et al.

observed hydrophobic interactions between hydrophobic domainsontheparticlesandthemucinfibers[20].Theseinter- actionsledtoanattractionbetweenparticlesandmucin.Similarly, Norris and Sinko observed that the amidine PS microspheres, which have the lowest hydrophobicity, also had the highest permeabilitythroughgastrointestinalmucin solution[7].Thus, to avoid lipophilic interactions, the particle surface must be hydrophilic,althoughotherpropertieshavebeenshowntohave aroleindiffusionand differenttypesofinteractionarebalance each other out.In the same study, the zetapotential was also shown to be a valuable indicator of the diffusion abilityof PS particles,withalowerzetapotentialfavoringahigherdiffusion ability. Repulsive electrostatic interactions were also observed between negatively charged particles and negatively charged mucin.However,ifaparticlewastooattractedbymucinbecause of lipophilic or electrostatic interactions, the particle became entangledin mucus. Bycontrast,if a particle wastoo repulsed by electrostatic interaction, it was unable to diffuse through mucus. In the case of hydrophilic particles, diffusion is easier forparticlesthathavenocharge(i.e.neutralparticles).

Dawsonetal.preparedcationicparticlesbyaddingacationic surfactant(PLGA-DDAB/DNA)toCOOHPS-particlestoenhance theirhydrophilicity.Theyobservedthatcationicparticlesaggre- gatedwithmucus,whichmighthaveledtolargermucusporesand promoted more rapid transport for a fractionof particles [12].

Muraetal.modifiedPLGAnanoparticlesurfacechargewithchit- osan(CS),pluronicF68(PF68),andpoly(vinylalcohol)(PVA)[24].

ApositivezetapotentialwasobtainedwithPLGA/CSnanoparti- cles,whereasitremainedalmostneutralforPLGA/PVAnanopar- ticles, and was negative for PLGA/PF68 nanoparticles. PLGA nanoparticlesexhibitedahydrophobicsurface,whichinteracted withthehydrophobicdomainsofthemucinchains.PLGA/CSand PLGA/PVA nanoparticlesbecame entrapped by mucus,whereas PLGA/PF68nanoparticlesdiffusedunimpededbetweenthemucin networks. Hydrophobic interactions were balanced by electro- static repulsions. The coating with Pluronic¹F127 (PF127) on PLGAparticlesledtoanear-neutralsurfacecharge,whereascoated particlesdiffusedmorefreelyinCRSMcomparedwithuncoated particles[30].ThemodificationofliposomeswithPF127improved diffusion through native rat intestinal mucus, because of the distribution of the hydrophilic polyoxyethylene part of PF127 on the liposome surface [14]. As a consequence, hydrophobic and electrostaticinteractions ofthe liposome withmucin were reduced.

CraterandCarriershowedsignificantdifferencesbetweenanio- nicandcationicparticlemucus-penetratingcapacities[8].Particle Probe nanoparticles (Non-mucoadhesive)

Native (Untreated) (b)

(a)

Synthetic nanoparticles

Mucin network Particle–mucin

Interaction Nanoparticles

Synthetic nanoparticles (Mucoadhesive) Sample trace of probes

Mucus mesh elements

Drug Discovery Today

FIGURE4

(a)Amucusdisruptioninducedbyparticles.Amucusfibernetworkis depictedontheleftwiththeintroductionofparticlesleadingtotheir entanglementwithmucusresultinginachangeofthefibernetwork.(Figure reproduced,withpermission,fromMcGillandSmyth[9].)(b)Thepotential effectsofmucoadhesiveparticlesonthemucusstructure.Mucoadhesive particlescanincreasemucusporesizesbybundlingmucinfiberswith adhesiveinteractions.(Figurereproduced,withpermission,fromWangetal.

[76].)

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mobilitywasinverselyrelatedtosurfacezetapotential.Sufficient surfacecoverageofanionicfunctionalitiesobtainedwithanionic (carboxylateorsulfate)particlessuppressedtheattractiveinterac- tions between the hydrophobic PS particle cores and mucin, whereasinadequatesurfacecoveragewithcationic(amine)parti- clesresultedinincreasedhydrophobicinteractionsandledtothe formationofparticleaggregate.Asaconsequence,negativecharge coveringresultedinasignificantlyhighertransportrateforpar- ticlesexhibitingahydrophobiccore.

Mucoadhesivemolecules

Asdiscussedabove,theliteraturehasdemonstratedevidencefora linkbetweenmucoadhesionandmucodiffusion.Tosomeextent, mucoadhesivenano-ormicroparticleshavemorepossibilitiesby whichtodiffuse,iftheymanagetoavoidbecomingentrappedin mucus becauseof interactions that aretoo strong. Thus, some studieshave usedmucoadhesivemolecules associatedwith col- loidalcarrierstoimproveparticlediffusionthroughmucus.Var- ious novel mucoadhesive polymers have been developed, includinglectins,thiolatedpolymers,bioadhesivenanopolymers, pluronics, alginate-polyethylene glycolacrylate and poloxomer [94].Forexample,Ezpeleta etal. showed thatlectinconjugates used for the delivery of hydrophobic drugs had an important affinityformucin[73].CSisanotherwell-knownmucoadhesive polysaccharide that forms disulfide bonds with cysteine-rich domains of mucus and also displays electrostatic interactions.

Mucoadhesioneffectiveness depends on polymer chemicalfea- tures,suchasMW,chainlength,spatialarrangement,flexibility, hydrationofpolymer, hydrogenbonding,charge,and polymer concentration. Moghaddam et al. found better mucoadhesion withsmallernanoparticleswithmediumMWCS[95].Thispoly- merwasmodifiedbytheadditionofthiolgrouptoobtainthio- latedpolymerscalled‘thiomers’,capableofformingathiolsulfide exchangereaction.Bravo-Osunaetal.developedamodifiedCS, calledthiolatedCS,whichleftparticlesstillabletodiffusethrough themucus[77].Gradaueretal.alsofoundthatthiolatedCS-coat- ing doubled liposome mucoadhesion compared with uncoated liposomes[96].Thedesignofnanomicellesbasedontheacetyl- cysteine(NAC)functionalizedCS-vitaminEsuccinatecopolymer exhibitedanabilitytopenetratemucus[83].Asa consequence, NACmoleculesincreasedthebioavailabilityofCS-vitaminEsuc- cinatenanomicelles,becauseofitsgoodthiolactivity.Petitetal.

alsofoundthatCSenhancednanoparticlemucoadhesion.More- over,themucoadhesionwasenhancedtwofoldbytheintroduc- tionofthiolgroupsonthesurfaceoftheCSnanoparticles[48].A novel preactivated thiolated CS improved mucoadhesion com- paredwiththiolatedCSbecauseofmoreactivesulfhydrylmoieties beingavailablethatprotectedthiolagainstearlyoxidation[71].A novelamphiphiliccopolymer wasdevelopedbyWang etal.by grafting glyceryl monostearate on CS [84]. This hydrophobic modificationincreasedthemucoadhesionoftheCSnanoparticles significantly(P<0.05).Chenetal.comparedthemucuspenetra- tionofliposomesmodifiedwithPF127orCS[97].Theydemon- stratedthatPF127-liposomeswereinclinedtopenetratethemucus andthentoaccumulatemoreeffectivelyinintestinaltissue,owing totheirmoreneutralandhydrophilicsurfacecomparedwithCS liposomesandnon-modifiedliposomes.MostCSliposomeswere trapped in the mucus, resulting in limited mucuspenetration.

Moreover, CSuse isnotwithoutrisktothe administrationsite, becauseCShasa tendencyto formcomplexeswithmucin and other proteins, which could cause major disturbances to the epitheliummembrane[98].

Coatingparticlewith PEG

Toavoidhydrophobicandelectrostaticinteractions,mucus-pene- tratingparticles(MPPs)werecoatedwithPEG,ahydrophilicand uncharged polymer. Thiscoating minimized efficiently particle adhesiontomucusconstituents[30].Griffithsetal.observedthat non-ionicpolymers,suchas10-kDaMWor100-kDAPEGdidnot interactwithmucin,whereasdendrimersandpolyethylenimine (PEI) exposed strong electrostatic (pH-dependent) interactions.

Therefore, by designing polymer-based drug delivery systems, electrostaticinteractionscanbemodulatedtoobtaingooddiffu- sion through mucus [10]. Similarly, the results of Tang et al.

suggested that the sufficient PEG density of poly(sebacic acid) (PSA)–PEG particles providedrapid nanoparticle penetration of CVMandCFS[23].PSAparticleswerestronglytrappedbyCVM, whereasPSA–PEGparticlesdiffusedunimpeded.Laietal.showed thatcoatingwith2-kDAPEGchainsincreasednotonlyPSnano- particle transport rates in CVM, but also the homogeneity of transport[19].

In agreement with findings in CVM [19], a dense covalent coatingoflowMWPEGledtoparticlespenetratingmoreeasily inothertypesofmucus,suchasCFM[28],CRSM[30],andHAM [27]. Similarly, PEG-coated particles of 100, 200 and 500nm penetrated mucus more rapidlythan did uncoated particles of thesamesize[18].Zabaletaetal.comparedtheapparentperme- abilitythroughintestinalrattissues ofparticlescoatedwithdif- ferent size of PEG: 2, 6 or 10kDa MW [47]. The apparent permeability of particles pegylated with PEG of 2kDa MW or 6kDa MWwas 2.5 times higher than nanoparticles pegylated withPEGof10kDaMW.Alowerinteractionbetweenmucuslayer andnanoparticlespegylatedwithPEGoflowerMWexplainedthe findings.Wangetal.alsoincreasedthecoatedparticledisplace- mentsinCVMbyareductioninPEGfrom10kDaMWto2kDa MW [20].However, particles coated with 5kDa MWdisplayed rapidmucus-penetratingproperties.Theseresultsindicatedthata crucial MW threshold exists between 5 and 10kDa. A small differenceinthesurfacePEGcoverageledtoa700-timesdecrease in the transport rate of PEG 2kDA-PS particles with 40% PEG coverage,comparedwiththesameparticlecoveredwith65–70%

PEG.Mertetal.observedthatPLGA–vitaminE-PEG1kDanano- particleswereasstronglytrappedinCVMasuncoatedPSnano- particles,despitethecoating,whereasPLGA/vitaminE-PEG5kDa nanoparticles rapidly penetrated CVM. This was the result of inadequatesurfacecoverageof1-kDAPEG[25].Similarly,mucoad- hesionofDNAparticleswasnotreducedbylowMWPEGcoatings, probablybecauseofinadequatePEGsurfacecoverage[85].Thus, inadequatePEGsurface densityappearsto beacruciallimiting factorforthedevelopmentofMPP.Theseresultscompletedthe design requirement ofPEG-coatedMPP. Inconclusion,suitable particles mustexhibit:(i)PEGofsufficientlylowMWand(ii)a sufficientlyhighdensityofPEGsurfacecoverage.

ThehydrophobiccoreofPSparticlesformedpolyvalentadhe- sive interactionswithhydrophobicdomains alongmucinfibers and possibly with other mucus constituents. Coating particles

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withPEGmightreducetheseparticle–mucusadhesiveinteractions if the MWof PEG is toolow to support adhesionby polymer interpenetrationandhydrogenbonding[99–101].PEGwithlow MWadoptedabrushconformationthatcouldfacilitatethediffu- sionofparticlesinmucusbyhinderingthehydrophobicinterac- tions[99,100],as,forexample,isthecaseforPEGof2kDaMWor 6kDaMW.Bycontrast,thedispositionoflongerPEGchains(i.e.

10kDa)wasdifferentatthenanoparticlesurfaceandfavoredthe interpenetrationandinteractionwiththemucusfibers[101].PEGs withatoolowMW,forexample1kDa,weredistributedinsideor physically adsorbedon the nanoparticlesurface. The pegylated nanoparticles obtained had a conserved high affinity for the mucus [99]. Regardless of these considerations, biodegradable MPPshave been developedandtested invitro[102] and invivo [57].However,rapidpenetrationofCVMbytheseMPPswithPEG MWfrom1kDato10kDashowedthatalargerangeofPEGMW canallowthepreparationofmuco-inertnanoparticles.Thedeter- minationofPEGMWrangecanbeaffectedbyvariousfactors,such astheparticlesize,corematerial,typeofmucusandsurfacePEG density.MPPsimprovedmucusdiffusion,vaginaldrugdistribu- tion and retention without causing inflammation, and PEG improvedthe mucuspenetrationofothercarriers,suchassolid lipidnanoparticles[103].

Diffusionenhancer

McGillandSmythtreatedmucuswithfunctionalizedPSnano- and microparticlestodisruptthe mucus beforemolecule dif- fusion[9].Thedisruption wassignificant,increasingpermea- tion of fluorescein and rhodamine through different mucus models.Similarly,Wangetal.fromtheteam ofJ.Hanesused highconcentrationsofmucoadhesiveparticles(MAP)(Fig.4), which enlarged mucus mesh pores to increase muco-inert PEG-coated particlediffusion bytenfold[76].Themucoadhe- sive properties of amine-modified PS particles sized 200nm resulted from the hydrophobic core and positive charges.

Exposure toMAP canbe a dangerous strategy becauseit can significantly increase the risk of infection or toxicity by

enhancingpenetrationbypathogensorotherforeignparticles withmuco-inert surfaces. Ensign showedthathypotonic for- mulationsimproved epithelialsurfacedistribution andreten- tion of MPP [56].

Anotherstrategytodisturbthemucuslayeristousemucolytic molecules,suchasDNaseandN-acetylcysteine,toenhancenano- particlediffusionthroughCFS[104].Mu¨lleretal.functionalized particleswithpapain,ahighlymucolyticenzyme,toreducemucin crosslinks[105].Asaconsequence,theapplicationofthesepar- ticles onPIM decreased mucus viscosityand improved particle diffusion.

Concluding remarks

Diffusion isa complexphenomenonthat is sensitiveto mucus composition and experimental parameters. To predict in vivo reality,the mucusselectedforinvitrostudiesmustbesimilarin compositionandstructuretotheinvivotargetedmucusandthe experimentalparametersmustbecontrolledcarefully.

Mucusis anefficient barrier forparticle diffusionbecause of physicochemical (hydrophobic, electrostatic, and hydrogen) interactions and steric occlusion related to its structure. Three strategies to improve diffusion through mucus have been describedin the literature: (i) disruption of the mucus barrier;

(ii)adhesiveparticles;and(iii)MPP[69],theimportanceofwhich hasincreasedoverthepastfewyears[106].

InthisFoundationReview,wehaveshownthattheresultsfor diffusionstudiesareoftenlinkedtothemodelused;therefore,a standardexperimentalprotocolisneededtoenablecrosscompar- isonofthecolloidsintermsoftheirabilitytodiffuseacrossmucus.

Theset-upofpredictivemodelsisalsomandatorytoenablethe designofeffectivecolloidalcarriersthatwilldiffuseeasilythrough mucus,thusimprovingtheperformanceofthesenewdrugdeliv- erysystems.

Acknowledgements

A-C.G.isafellowfromEthypharmsupportedbyAssociation NationaledelaRechercheetdelaTechnologie(ANRT).

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