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Teaser Nanoemulsions are among the most promising future drug carriers with remarkable potential for the brain delivery of a wide variety of drugs.

Nanoemulsions in CNS drug delivery:

recent developments, impacts and challenges

Zahra Karami

¹

, Mohammad Reza Saghatchi Zanjani

¹

and Mehrdad Hamidi

¹^,2

1ZanjanPharmaceuticalNanotechnologyResearchCenter(ZPNRC),DepartmentofPharmaceutical Nanotechnology,SchoolofPharmacy,ZanjanUniversityofMedicalSciences,Zanjan,Iran

2TritaResearchCenter,Zanjan,Iran

Despite enormous efforts, treatment of CNS diseases remains challenging.

One of the main issues causing this situation is limited CNS access for the majority of drugs used as part of the therapeutic regimens against life- threatening CNS diseases. Regarding the inarguable position of the nanocarrier systems in neuropharmacokinetic enhancement of the CNS drugs, this review discusses the latest findings on nanoemulsions (NEs) as one of the most promising candidates of this type, to overcome the challenges of CNS drug delivery. Future development of NE-based CNS drug delivery needs to consider so many aspects not only from a

physicochemical point of view but also related to the biointerface of these very small droplets before achieving clinical value.

Introduction

Neurologicaldisorderscontributeto~11%oftheglobalburdenofdiseaseandthisnumberis anticipated to rise in the coming years owing toan aging global population. Nowadays, significantresearcheffortshavebeendevotedtothedevelopmentofimprovedtherapiesfor central nervoussystem (CNS)diseases.However, despite tremendousefforts,treatment of CNSdiseaseshasbeenlimitedbytheinabilityofthemajorityofneuroactivedrugstoreach CNScompartmentsinsufficientamounts[1].Manypotentialdrugs,despitebeingeffective at thedesirablesiteof action,havebeen discardedthroughclinicalstudiesbecause ofthe limitedentrytothebrainparenchyma.Twophysiologicalandbiochemicalbarriers[i.e.,the blood–brainbarrier(BBB)andblood–cerebrospinalfluidbarrier(BCSFB)]areresponsiblefor thelimitedCNSaccessbythetherapeuticagents[2].TheBBBandBCSFBareconceptualized bythe brainmicrovessel endothelial cells(BMECs) andtheepithelial cellsof thechoroid plexus,respectively.TheBBBis,infact,a specializedstructurethatcontrolsthepassageof materials from the systemic circulation into the very sensitive and vital CNS tissues.

However,it alsoexcludesvarious pharmacologicallyactiveagents frombeing availablein an effectiveamount.

ReviewsKEYNOTEREVIEW

ZahraKaramireceived herBScinchemistryfrom TehranUniversity,anMSc inanalyticalchemistryfrom KharazmiUniversityand herPhDinpharmaceutical nanotechnologyfrom ZanjanUniversityof

MedicalSciences.DrKaramiisanassistantprofessor ofpharmaceuticalnanotechnologyinZanjanSchoolof Pharmacy,Iran.Herresearchinterestsfocusonthe design,preparationandevaluationofvarious functionalnanomaterials(includinglipid-based nanoparticles,nanoemulsions,liposomes,polymeric nanoparticlesandironoxidenanoparticles).Todate, shehaspublishedseveralarticlesinpeer-reviewed journals.

MohammadReza SaghatchiZanjani receivedhisdoctorateof veterinarymedicine(DVM) fromtheIslamicAzad University,TabrizBranch,in 2017.HeattendedZanjan UniversityofMedical Sciencesasaresearcher

underthesupervisionofMehrdadHamidiandHamed Ghavimifrom2016to2018.Heiscurrentlya researcheratTritaPharmaceuticalCompany,Zanjan, Iran.Hisresearchinterestsfocusonanimalstudies includingsurgery,pharmacokineticsandbehavioral studies.

MehrdadHamidi obtainedhisPharmD(1993) andPhD(1999)degrees fromTehranUniversityof MedicalSciences,Iran.He subsequentlycarriedout twopostdoctoral fellowshipsatUniversityof Alberta,Canada(1999˘

2001)andUniversityofToronto,Canada(2001˘2003).

DrHamidiisaprofessorofpharmaceutical nanotechnologyatZanjanSchoolofPharmacy,Iran.

Hismainresearchfieldofinterestisnanotechnology- baseddrugdeliverysystemsfocusingonBBB transportofCNS-activedrugs.Hehaspublished severalarticlesinwell-knownjournals,amongwhich twopaperswereselectedasthetopfiveworld’s most-citedpapersin2009and2007.Hehasalso publishedsevenchaptersininternationalbooksand fiveUSpatentsinnanotechnology.DrHamidiis amongtheworld’stop1%ofscientistsbyESIranking, since2013.HeisanEditorialBoardMemberof13 internationaljournals.

Correspondingauthor:Hamidi,M. ([email protected])

1359-6446/ã

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Despitethis worryingpicture, significant evolution by intro- ducingnewdrugdeliverysystems(DDSs),withtheaimofeffective drugtransporttotheCNS,havebeenreportedwithmanyprom- isingresults.Amongothers,variousnanotechnologyDDSshave beenexploredascarrierstobypasstheBBBefficiently[3,4].The presentreviewwillprovideinsightsintothepossibleapplications ofnanoemulsion(NE)-basedsystems,oneofthemostpromising types ofnanocarriers, for the enhancement ofCNS delivery of bioactiveagents intended to find new solutions fortherapy of hard-to-treatCNSdisorders,particularlybraincancersandneuro- degenerativedisorders.

Biological aspects in CNS drug delivery

Blood–brainbarrier

TheBBB, the key barrier betweenthe CNS and the peripheral circulation,isthe homeostaticdefensemechanismofthebrain that regulates the traffic of immune surveillance cells (macrophages),xenobioticsandendogenouscompoundsinandoutof theCNS.ThefunctionalcomplexityoftheBBBiscausedmostly byseveralbiochemical-regulatingmechanismsexpressedincere- bralendothelial cells and the presenceof the specialized tight junctionstructures betweenthem. In particular,tight junction proteins(i.e.,claudins,occludin)andadherentjunctionproteins (i.e.,junctionaladhesionmolecule)createthehightransendothe- lial electrical resistance (>1500

V

^cm² ^compared ^with ^3–33

V

cm² of other organs) that limits the transport of hydrophilic molecules[5,6].Theyarelinkedtotheactincytoskeleton,thereby forming the most intimate cell–cell connection. BMECs have severaluniquestructuralfeaturessuchastheabsenceoffenestra- tionsandverylimitednumbersofpinocytoticvesicles;butthey possessmoremitochondria.Inaddition,thereareotherobstacles againstthepassageofsolutesacrossBMECs,includingdegrading enzymes:ecto-andendo-enzymes[7].Thesemorphologicaland functionalfeatures,collectively,resultinlimitedtranscytosisand endocytosiswhilesupportingthelowpermeabilityoftheBBBfor manysubstances. Thereareother additionalstructures suchas pericytes, astrocytes and a discontinuous basal membrane (or basallamina),assistingBMECsintheirspecializedbarrieraction.

Thepericytesareadjacenttothebraincapillariesalongthebasal laminaandplayimportantpartsintheregulationofCNShomeo- stasis, BBB integrity, macrophage activity and modulation of bloodflow[8].Thebasallaminaissituatedbetweenbraincapillar- iesandthepericytesandconsistsoflaminin,fibronectin,tenas- cin, collagens and proteoglycan. It serves as an extracellular matrixprovidingascaffoldforcellmigration,mechanicalsupport for cellattachment and separation of adjacent tissue. Another importantsupportivecell-typeinvolvedintheBBBfunctionare astrocytes,whichentirelyenvelopBMECsandpericyteswiththeir end-feet.Recentstudieshaverevealedthatastrocytesareimpor- tantinmodulatingtheBBBfunctionbyregulationofwaterand ion homeostasis and secretion of chemokines as a sensor of pathologicchanges.

Blood–cerebrospinalfluidbarrier

TheBCSFBbarrier,known asthe secondarybarrieragainstdrug penetrationintotheCNS,iscomposedofchoroidplexusepithelial cells. The choroid plexus consists of a highly vascularized,

‘cauliflower-like’structurewithfrequentvillithatprojectintoall

fourcerebralventricles[9].IncontrasttotheBBB,thecapillariesof the choroid plexus arefenestrated and noncontinuous withan obstructionsurroundingthemmadebyamonolayerofpolarized epithelial cellslinkedtogether bytight junction proteins.These tightjunctionsareamajorobstacleagainstpassageofmostmacro- molecules and ionized materials. The cerebrospinal fluid (CSF) secretedfromthechoroidplexusepithelialcellsfillstheventricles ofthebrain,thespinalcanalandsubarachnoidspace.CSFsecretion is~350

m

^l/minⁱⁿânâdult^male,^which^representsâ^turnover^rateôf

~0.4%min¹.ThesecretionofCSFmaintainstheconcentrationof substancesthatcanpenetratethebrainviapassivediffusioninan extentlowerthanplasma–aphenomenonknownastheCSFsink effect. The sink effect isgreater for large molecularweight and hydrophiliccompounds.Inaddition,thepresenceofionchannels, transportersandreceptorshasbeenreportedattheBCSFB[10].The penetrationofadrugmoleculeacrossthesetwobarriersdependson severalphysicochemicalpropertiesmainlyincludinglipophilicity, sizeanddegreeofionization.

Pathways acrossbrainbarriers

Ingeneral,penetrationofmaterialsintothebraincanbeclassified intotwo maincategoriesofparacellularandtranscellularpaths.

Onlysmall hydrophilicmaterialscan diffusethroughthe tight junctionsbutnottoahighdegree.However,owingtotheunique propertiesofthetightjunctions,paracellulartransportofhydro- philic drugs is virtually absent and transcellular transport by passivediffusionisonlyavailabletomoleculesthatfulfillcertain criteriasuchas:molecularweight<500Da,logP2,beingnon- ionizedandcumulativenumberofH-bonds<10[11].Obviously, justasmallproportionoftherapeuticshastheseconditions.By contrast, ithas been foundthat some ofthe nutrients suchas bloodglucose,proteinsandpeptidescaneasilycrosstheBBB[12].

Aseriesofmembranetransportersystemsareexpressedinthe luminaland/orabluminalsurfaces(i.e.,blood-to-brainandbrain- to-blooddirections)oftheBBB.Thesetransportersareresponsible forthedifferentinfluxoreffluxbehaviorsoftheBBBfordifferent substances exposed to this barrier.In addition, thereare some carrier-mediated endocytosisand exocytosismechanismsin the BBBandBCSFB.Incarrier-mediatedendocytosisthesolutesbind tospecificmembraneproteincarriers,thenallowcellpenetration via endocytosis. Mechanistically, a conformational transforma- tioninthecarrierproteintakesplacewhichprovidesthepossibili- tyforthesolutetopasstotheothersideofthecellularmembrane.

The energyformovementofmoleculesagainsttheirconcentra- tiongradientissuppliedbyATP.Themaintwoclassesofendocy- tosisarefluid-phaseendocytosis(usuallyreferredtoaspinocytosis) andsolid-phaseendocytosis(knownasphagocytosis)[13].Fluid- phaseendocytosisisa nonspecificuptakeofextracellularfluids, driven by theconcentration ofthe extracellular side.Although thistypeofendocytosisoccursataverylowlevel,thismechanism is assumed to be noncompetitive becausethere is no needfor ligandbinding.Receptor-mediatedtranscytosis(RMT)providesa meansforselectivebrainuptakeofsmallandlargemoleculessuch ashormones,growthfactors,enzymesand plasmaproteins. Be- causeofthelimitedquantityofthereceptorsontheBMECsurface, RMT is, unfortunately, a saturable mechanism. RMT has been extensivelystudiedforbraintargeting.Adsorptive-mediatedtrans- port(AMT)isinitiatedbyanelectrostaticinteractionbetweena

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positiveingredientandthenegativelychargedplasmamembrane surface.IncontrasttoRMT,AMTshowsloweraffinityandhigher capacity.Macromoleculesareconcentratedbeforetransportation by interactionwith the BMECsurface,mainlyvia the clathrin- mediatedmode.TheadventofvariousCNSdrugdeliverytechnol- ogiesfocusesonAMT,usuallybyexploitingthebenefitofcationic proteins or cell-penetrating peptides. Moreover, cell-mediated transcytosis is an effective point of access forsome pathogens for instance Cryptococcus neoformans and HIV into the brain, known as the ‘Trojan horse’ model. This mechanism depends on immune cellslike monocytes or macrophages to reach the other side of the intact BBB [14]. Unlike the aforementioned transportpaths,cell-mediatedtranscytosiscanbeappliedvirtually foranykindofmoleculesaswellasparticulatesystems.

Nanoemulsions

NEsarecolloidaldispersionsconsistingoftwoimmiscibleliquids stabilized by surface-active agents, with dispersed droplets of diametersintherangeof20to500nm(Figure1)[15].Insome publicationsNEsarealsoreferredtoasmini-emulsions,ultrafine emulsionsor submicronemulsions.Becauseoftheirsmallsizes, NEs areconsideredtobekineticallystablesystems, becausethe Brownianmotioneffectsdominategravitationalforces,thuslead- ingtohigherresistanceagainstdropletaggregationthanconven- tionalemulsifiedsystems.ItisimportanttodifferentiateNEsfrom microemulsions, in that the second one, being anequilibrium structure,ishighlysensitivetoanyvariationsintemperatureand chemicalcomposition.

Furthermore,ionicornonionicsurfactantsarefrequentlyused intheNEstructuretopreventflocculationsviaelectrostaticand stericstabilization[16].ThemainmechanismofinstabilityofNEs isreportedtobetheOstwaldripening.Becauseofthedifferencesin Laplace pressure, the smallerdroplets ofNEs becomegradually smallerinsizeuntilbeingdissolvedordisappearingintheexternal phase.Thelargeonescontinuetogrowandfinallyseparateout, affectingthelong-termstabilityofthedispersion.

Dependingonthedispersedphasediameterandaccordingto theappearance,NEsareclassifiedintotwogroups:transparent ortranslucent(50–200nm);andmilky(upto500nm).Inaddi- tion,depending oningredients, internalandexternalphases, therearetwomaintypesofNE,referredtoasbiphasic[i.e.,oil- in-water(O/W)orwater-in-oil(W/O)]andmultipleNEs(W/O/

W). The relative volumes of internal and external liquids, termedasphase:volumeratio(

F

^),^determine^NE^droplet^num-

beraswellassystemstability.Generally,theinternalphaseis thephase presentin a lowervolumein comparisonwith the continuousphase,whereincreasing

F

>40%canresultinphase inversionofaW/ONEtoanO/Wone.AmultipleNEcontainsa dispersedphasethat,inturn,includesanotherinternaldroplet ofadifferentimmiscibleliquidphase.

SomeexcellentpropertiesofNEssuchasthecolloidaldispersion ofthehydrophobictherapeuticagents,cellulartransporteitherby paracellularortranscellularroutes,preventingtheentrappedsub- stancesfromhydrolysisand/orenzymaticdegradationand,finally,theirremarkablebiocompatibilitymakethemhighlypromising carriersforadvanceddrugdelivery[17,18].Additionally,incon- tactwiththecontinuousphase(water),oncethedrugdiffusesout oftheoildropletsitundergoesnanoprecipitationand,according totheNoye–Whitneyequation,thistransformationincreasesthe surface area of the drug leading to the highly accelerated and enhanceddrugdissolution.Furthermore,therateand extentof drugreleasecanbeeasilymodulatedbyvaryingthecomposition of nanodispersion to obtain a sustained or controlled release system.Moreinterestingly,theverylargeinterfacialareaprovides opportunitiesformore-advancedengineeringtodeveloptargeted DDSstospecificsites.Sometypicalexamplesofdrug-loadedNEs aimedatbraindelivery,relatedtothescopeofthecurrentstudy, aresummarizedinTable1.

Componentsofananoemulsion

AtypicalNEiscomposedofatleastthreeessentialcomponents:

oil,waterandanemulsifier,atappropriateratios[19].However,

Water in oil (O/W) nanoemulsion

Emulsifier

Dissolved drug molecules

Oil

Hydrophilic head

Hydrophobic tail

20-500 nm

Drug Discovery Today

FIGURE1

Schematicrepresentationofanoil-in-waternanoemulsionsystem.Nanoemulsionsarestablecolloidaldispersionscomposedoftwoimmiscibleliquids,withthe helpofsurface-activeagents:‘emulsifiers’.Generally,thedrugisdissolvedintheinternalphaseofnanoemulsions.

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theinternalandexternalphasescancontainothermultipleingre- dients.Thesolubilityofthedrugintheinternalphase(oil)isan essentialcriterionforselectionofoilsinO/WNEs.Edibleoilsand theirre-esterifiedfractions,generallyreferredtoassemisynthetic medium-chainderivatives[longchaintriglycerides(LCT),medium-chaintriglycerides(MCT)orshortchaintriglycerides(SCT)]

arefrequentlyusedtoprepareNEs.D-

a

-Tocopherol,oleicacidand ethyloleate havealso beenusedin avariety ofpharmaceutical NEs.

Emulsifiersareamphiphilicmoleculescapableofstabilizingthe NEsbydecreasingtheinterfacialtension(thesurfaceenergyper unitarea)to below10dynes/cmbetweenthe oily and aqueous phasesofthedispersion.Besides,theyplayavitalpartinstabiliz- ing NEs through repulsive electrostatic interactions and steric hindrance [20]. Usually, a surfactant is used as an emulsifier, butamphiphilicproteins,polysaccharidesandlipidscanalsobe appliedintheformulationofNEsasastabilizer.Anappropriate emulsifiershouldbeadsorbedeasilyaroundtheinternaldroplets toformacompleteandcoherentfilm,therebybeingeffectiveata fairlylowconcentrationfortheconstructionofthedesiredsizeof droplets,andalsotooptimizethezetapotentialandviscosityof thesystem.Itiscommonlybelievedthatsurfactantswithalow hydrophilic–lipophilicbalance(HLB)valueof3–6arefavoredin

preparationofW/ONEs,whereasemulsifierswithhighHLBvalues (8–18) are ideal for O/W types. Some common emulsifiers employedintheformulationsofpharmaceuticalNEsarelecithin (phosphatidylcholinederivedfromeggyolkorsoybean),Cremo- phor¹EL(polyoxyl-35castoroil),Tweens¹(polyoxyethlenesor- bitanmonolaurate),Spans¹(sorbitanmonolaurate),poloxamers, sodiumdeoxycholate(bilesalt),casein,gums,starchderivatives andblockcopolymers[21–23].

Methods ofpreparationofnanoemulsions

NEsarethermodynamicallyunstableowingtoapositiveGibbsfree energyoftheirformation.Therefore,preparationofNEsisnota spontaneous process,thus needingenergy alongwith applying emulsifiersto stabilize the formednanodroplets[20].Thereare typicallytwoapproachesforproductionofNEs,classifiedashigh- energyemulsificationandlow-energyemulsification.High-energy proceduresinvolveapplyingamechanicaldevicetocreategreat disruptiveforcestobreakdowntheinternalphase,thendispersing itintothecontinuousphase.Someofthemainmethodsofthis group are: stirring, ultrasonication, high-pressure homogenization,microfluidizationandmembraneemulsification[24,25].In low-energymethodsoilnanoglobulesstarttogrowviachanging the conditionofamixed oil-water-surfactantsystem.The latter TABLE1

Examplesofnanoemulsionsintendedforbraindelivery

Drug Composition Method Size(nm) Outcomes Refs

Carbamazepine Soybeanoil/

Lecithin/1-O- alkylglycerol

High-pressure homogenization

207.30.8 Increasedthebrainavailability2.37timescomparedto freedrug

[93]

Paclitaxel Pine-nutoil/Lipoid^TM Sonication 212.51.6 Increaseincytotoxicitycorrelatedwithenhancementin apoptoticactivityinU-118cellstreatedwith

nanoemulsionofcombinationofpaclitaxelandceramide [94]

Atovaquone Grape-seedoil/ Polysorbate80

Spontaneous emulsification

203 NE-ATefficiencywasprovenbyincreasingsurvivaltime anddecreaseparasiteloadininfectedmicewithT.gondii, RHstrain

[83]

Curcumin Linseedoil/Lipoid^TM/ Polyethyleneglycol

Highpressure homogenizer

10311 Improvementoftotalantioxidantactivityinbrainabout sixtimesincomparisontocurcuminsolutioninlinseedoil

[95]

Carbamazepine Soyaoil/

Tetrahydrofuran

Sonication 95 Significantanticonvulsantactivityagainstseizuresand relativebioavailabilityof160%comparedwith unmodifiedsubstance

More-effectivelypenetratedthroughBBBby1.5times relativetounmodifiedsubstance

[96]

Curcumin Labrafac¹lipophile WL1349/Solutol¹ HS15/Transcutol¹

Pseudoternaryphase diagram

676 IC50of16.41m^MônÛ87^cells^compared^to^24.23m^Môf

curcuminsolution

[97]

GinkgolideB Soybeanoil/

Lecithin/Ethyllactate

Magneticstirring 80–100 Memoryimprovementofratswithcognitionimpaired wasconfirmed

Significantlyinhibitedacetylcholinesteraseactivityand enhancedtheactivityofcholineacetyltransferaseinthe hippocampus

[98]

Docetaxel Soybeanoil/

Lecithin/Poloxamer 188/Glycerol/Cholic acidsodium

High-energy homogenization

72.3 InvivotoxicitysignificantlylowerthanTaxotere¹ Abilitytotargetgliomasitesandprolongthemedian survivaltimeofmicewithgliomas

[99]

Diazepam Medium-chain triglycerides/

Soybeanoil/

Lecithin/Tween¹80

High-pressure homogenization

195–220 Fastandintenseinitialdistributionintoratbrain, suggestingtheirapplicabilityinurgentsituations

[100]

Resveratrol Orangeoil/Grape- seedoil/Tween¹80

Magneticstirring 86.412.7 Inducedneuroprotectivebenefits

Showedpreventivepotentialforpostoperativecognitive dysfunctionviatheSIRT1signalingpathway

[101]

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methodhasdefinitephaseinversiontemperatures,emulsionin- versionpointsandspontaneousemulsificationconditions[26,27].

Main advantages of nanoemulsions in CNS drug delivery

Impactondrugsolubility

Solubility in biological environments is one of the important factors in meeting the therapeutic role of active substances.

AccordingtotheBiopharmaceuticalClassificationSystem(BCS), ClassIIandClassIVhaveasolubilitylimitationinaqueousmedia.

Forthisreason,theyarenotabletofulfilltheirtherapeuticactivity inlowconcentrations,thusneedinghigherdosestoachievean appropriatepharmacologicalactivity[28].BCSClassIIdrugsdo nothaveaparticularproblemintermsofmembranepermeability withtheonlycrucialissuebeingtheiraqueoussolubility,which canbeimprovedbyusingsolubility-enhancementtechniques–in particularthe useofnanocarriers.BCSClassIVdrugs,however, sufferfromalimitedpermeabilityacrossbiologicalmembranesin additiontotheaqueoussolubilityproblem.Therefore,toimprove thebiologicalfunctionsofthiscategory,itisnecessarytofocuson increasingsolubilityandpermeabilityatthesametime.Giventhe complexstructureoftheBBBmentionedearlier,thechanceofa substanceenteringthebrainislimitedbyphysicochemicalaswell asbiochemicalconstraints,therebyfavoringfat-lovingsubstances withamolecular weightof~500Da orlower.Therefore,itseems thattheuseoflipophilicnanocarriers,likeNEs,couldbeagood option for loading lipophilic and amphiphilic drugs with BBB penetration problemstodeliverthem tothe brain.Bycontrast, besides thehighloadingcapacity, they canbeusedastargeted drugdeliverycarriersfortheCNSactivetherapeuticsowingtothe possibilityofparticleengineeringandsurfacemodification.Sofar, severalstudieshavedemonstratedtheefficacyofNEsinincreasing thesolubilityofvariousinsolubledrugs[29,30].Itcanbeconclud- edthat,byincreasingthesolubilityofthedruginthebloodand the target tissue, itstherapeutic function canbe improved.In- creasingthesolubilityofthepoorly-water-solublematerialsloaded intheNEsisdonebytheconsiderablefractionalvolumeoftheoily phaseinthestructure,thehighsurfaceareaandthepresenceofan activefilmlayerofsurfactantsattheinterfaceoftheinternaland externalphases.

Ferreiraetal.developedketoprofen-loadedNEs(KP-NEs)stabilized by pullulan asan antiglioma formulation [31].The main ingredients used in preparation ofKP-NEs were Span¹80 and pomegranate-seed oil.The invitro releaseexperiments revealed thatnanoemulsionimproveddrugreleaserelativetothefreedrug solution, where~95% of drug was released in 5h. The authors proposed that enhancing the solubilityof the drug could lead to an increase in its plasma concentration and, subsequently, could improve the net brain permeation. KP-NEs showed 40%

inhibition of cell growth in incubation against a C6 cell line.

AnotherNE-baseddrugdeliverysystemwasdevelopedfromcastor oil, soybean lecithin and Polysorbate 80(P80) for intravenous administrationofthalidomide(THD)[32].Theinvitrodissolution profileshowedareleaseprofilesimilartothatofaTHDacetonitrile solution,with~95%THDbeingdissolvedwithin4h.Finally,the pharmacokinetic(PK)simulationoftheintravenous(i.v.)infusion ofdevelopedNEscontaining0.01%THDhasindicatedthatadose aslowas25mgcanproducetherapeuticplasmaconcentrations.In

anotherstudy,itwasobservedthat,byreducingthemeandiame- terofaNEincorporatedbyCoenzymeQ10(CoQ10),thecumula- tivereleasepercentageoflipophilicdrugwasincreased[33].Also, theNEformulationofCoQ10wasmoreefficientinenhancement ofdrugreleaseinphosphatebuffer(pH6.8)comparedwithfree CoQ10suspension.ThePKstudyinalbinoratsshowed1.81-times improvement in bioavailability compared with CoQ10 suspension. Moreover, the haloperidol-challenged rats treated with CoQ10 NEs revealed improved behavioral activities compared withthecontrolgroup,byreducingnigrostriataldopaminedeple- tion,thereforetreatingParkinson’sdisease.

Curcumin(CUR),alipophilicmoleculewithalogPvalueof3.28, haslimitedsolubilityinaqueousmedia.Soodetal.developedCUR- loadedNEs(CUR-NEs)madeofa mixtureofCapmul¹MCMand Captex¹500astheoilphase,Cremophor1ELandP80assurfactants, andPEG400andTranscutol¹asco-surfactants[34].Thecumulative releaseofdrugfromCUR-NEsinsimulatednasalfluidcontaining1%

SLS(sinkconditions)wasremarkablyhigher(P<0.001)incompari- sontothefreeCUR,wherethetotalCURreleaseoffreedrugandCUR- NEswere50.681.04%and74.341.3%,respectively.

Impactonmembraneinteractions

DifferentpathwaysaredescribedfortransferringdrugbyNEsinto thecellswithin thetargettissue (Figure 2).Because mostdrugs loaded into NEs are lipophilic, the main possibility for drug deliveryseems tobethroughthe fusionof theoily phase with thecell membranephospholipidsbylipid exchange.Therefore, NEs,becauseoftheoilphaseandthesurfactantlayer,seemtobea suitableoptionfortransferringCNSactivedrugs viainteraction withthe membraneofthe BMECs. Inaninterestingstudy,the braindelivery capabilityof somelipid-basednanostructures in- cluding solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) and NEs was investigated as sulforhodamine B vehicles[35].Evaluationofthedyeaccumulationinratsshowed thattheretentioninthebrainwasmost prolongedusingNEs– from20to50min.Itseemsthat,owingtotheflexibilityofNEoil droplets, compared with the more rigid nanostructured lipid- basedcarriers, the efficiencyofovercoming the BBB via carrier uptakeintothebrainisimprovedusingtheNEs.

Moreover,someofthepolyunsaturatedfattyacids(PUFAs)such asomega-3andomega-6possessspecialtransportersonthelumi- nalmembraneoftheBBB. UsingPUFA-richoil NEs,Vyasetal.

showedremarkableincreasedoralbioavailabilityandbraindeliv- eryofsaquinavir(SQV)–ananti-HIVproteaseinhibitor[36].By comparingNEformulationwithdrugsuspension,theC_maxand theAUC_0-1wereobservedtobefivefoldandthreefoldgreaterin thebrain,respectively,suggestiveoftheenhancedrateandextent ofdrugabsorptioninamurineanimalmodel.Theyalsostudied theroleofoiltypeinNE efficacyin braindeliveryofSQVina parallelstudy.ThebrainconcentrationofSQVfollowingadmin- istrationofSQV-loadedflax-seedoilNEsledtosignificantlyhigher (P<0.05)CmaxandAUC_0-1valuesascomparedwiththeCartha- mustinctorius(safflower)oilNEs.Thismightbeattributabletothe higherlevelofomega-3fattyacidinthe flax-seedoilrelativeto saffloweroil,where,flax-seedoilconsistsof58%omega-3and14%

omega-6,whereastheomega-6fattyacidcontentofsafflower-oilis

~75%.Thesefindings suggestgreatselectivityinthetransport of PUFAsacrosstheBBB.

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Sofar,varioustheorieshavebeendescribedtoexplainhowfatty acidspenetratethebrain.IthasbeensuggestedthatPUFAsbypass theluminaland the transluminalleafletsofthe BBB througha reversibleflip-flopmechanism.When thePUFAsreachtheneu- rons,acetyl-CoA synthetase traps them by creating acetyl-CoA thatdoesnothavethecapabilitytoexitthecell.Anothertheoryis basedonPUFA–albuminassociations,wherethePUFAstranslocate fromtheextracellulartointracellularleafletofthemembranevia transporters[37].Furthermore,PUFAsareexploredasactiveab- sorptionenhancersattheBBBbytriggeringmembranefluidifica- tion and modulation of the tight junctions; thus promoting paracellularandtranscellulartransportation[38].

Shah et al. showed that incorporation ofDALDA peptide in fatty-acid-richNEs significantlyimprovesbrainentryofpeptide comparedwith the free peptide solution,ultimately leadingto enhancedbrainexposure(3.3-foldhigherAUC_0-1)[39].Basedon theMRTvalues,DALDA-NEsprovidedhigherresidenceofpeptide inbrain(i.e.,145.4minvs198.4min)forfreeDALDAsolution.Ina similarstudy,theDALDA-C8peptide-encapsulatedomega-3-rich NE (DALDA-C8-NE) proved its ability in the capsaicin-pain-in- ducedfunctionalMRImodelinrats[40].Asignificant(P<0.05) differencewasdetectedinthepositiveblood-oxygen-level-depen- dentsignalinseveralbrainsectionsinvolvedinthepaincircuit,as characterized by the reduced extent of activation in animals receivingDALDA-C8-NEsbeforecapsaicinshock.

Impactonmodulationofdrug effluxtransporters

Despitetheentryroutes,thepresenceofaseriesofactiveefflux transports(AETs)ontheBBB,mainlyATP-bindingcassette(ABC) transporters,limitstheactivityofmanydrugsbyremovingthem

from the brain parenchyma back into the bloodstream [41].

P- glycoprotein (P-gp), multidrug-resistance protein (MRP) and breast-cancer-resistance protein (BCRP), the three important membersofthe ABCtransportergroup,aredemonstratedtobe responsiblefortheeffluxofawidevariety oftherapeuticagents such as methotrexate, vincristine, indinavir, camptothecins, anthracyclines,mitoxantrone,taxanesandcyclosporineA,among many others, from the brain parenchyma. It is obvious that inhibitionofBBBeffluxtransporterscanbeassumedasapossible strategyforimprovingbrainentryoftheirsubstrates.NEscanbe generallyconsideredasusefultoolsforbypassingtheP-gppump fromtwodifferentpointsofview:(i)thegeneral‘masking’concept oftheactiveagentsbythedropletsfrombeingdiscoveredbythe transporter while they enter the BBB; and (ii) the presence of surfactantssuchasP80inNEdropletsthathavebeenidentified aswell-knownP-gpinhibitors.Dorpevicetal.suggestedthatthe inhibition of the P-gp efflux system at the BBB by P80 and Poloxamer188(PL188)couldplayanimportantpartamongother features of P80- and PL188-containing NEs of risperidone to improvebrainavailabilityofthedrug[42].Indinavir,aprotease inhibitorusedinthetreatmentofHIVinfection,haslimitedentry tothebrainowingtoeffluxbyP-gpexpressedattheBBB[43].The pegylated NE of indinavir caused significantly enhanced drug concentrationsinbrainincomparisontothefreedrugsolution aswellasthecontrolblend[44].Theincreaseinindinavirlevelsin brain, despite the greater hydrophilic surface caused by PEG chains,couldbeexplainedbyprolongedcirculationtimeofstealth NEs, thereby leadingto the BBB penetration to a greaterlevel.

Further,ithasbeenreportedthatthePEGsandtheirderivatives inhibitintestinalP-gp;therefore,theotherprobablemechanism Blood

Brain

Lipid exchange

Transporter- mediated transcytosis

Receptor-mediated transcytosis

Adsorptive-mediated transcytosis

Transporters Transporter substrate

Receptor Targeting ligand

Nanoemulsion

Drug Discovery Today

FIGURE2

Proposedmechanismsofnanoemulsion(NE)transportacrosstheblood–brainbarrier(BBB).Lipidexchange:throughtheinteractionoftheoilphaseandthe surfactantlayerofNEswiththemembraneofthebrainmicrovesselendothelialcells.Transporter-mediatedtranscytosis:occursthroughtheinteractionofNEs withmembranetransportersexpressedintheluminaland/orabluminalsurfaces.Receptor-mediatedtranscytosis:aselectivebrainuptakeofNEsdecoratedwith targetingligandsthatbindBBBreceptors.Adsorptive-mediatedtranscytosis:initiatedbyanelectrostaticinteractionbetweenpositivelychargedNEsandthe negativesurfaceoftheBBB.

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wouldbetheinhibitionofP-gpattheBBBbyPEGasachemical entity.

Recent trends to optimize nanoemulsion application for CNS drug delivery

Intranasaladministration

Intranasal administrationhasbeen consideredasa noninvasive deliveryoptionthatbypassestheBBBandenablesdirectaccessofa varietyoftherapeuticagentstotheCNS,avoidingdrugfirst-pass eliminationbytheliverand gastrointestinaltract,orenzymatic degradationin plasmabeforereachingthe CNS.However, poor absorption, enzymatic degradation and poor permeation and retention of drugs can beaddressed as somedrawbacks of this route.Inrecentyears,NEsattractedagreatdealofattentionfor intranasaldrugdelivery,whichcanbeattributedtotheiradvan- tagessuchastheabilitytoprotectthedrugfrombiologicaland/or chemicaldegradation,thepotentialtoimprovethedrugabsorp- tion via inducing mucoadhesion or create a positive electrical surfaceforincreasingthe nasalresidencetime[45].To visualize brainuptakefollowingintranasal(i.n.)andi.v.administrationsof technetium-labeledrisperidonenanoemulsions(RP-NEs),agam- mascintigraphystudywasused[46].Aremarkablyhigherradio- activity accumulation was seen in brain following i.n.

administration ofRP-NEscomparedwithi.v.administration.In asimilarstudy,theolanzapinemucoadhesiveNEs(OZ-MNEs)and olanzapineNEs(OZ-NEs)werepreparedandtheirbiodistributions inratsfollowingi.n.andi.v.administrationwereexamined[47].

From aninvivostudy, thebrain-to-blood uptakeratiosof0.45, 0.88,0.80and0.04wereobtainedfordrugsolution(i.n.),OZ-NEs (i.n.), OZ-MNEs (i.n.) and OZ-NEs (i.v.), respectively, at 0.5h.

Gammascintigraphyimagingoftheratbrain,bycontrast,showed a rapidand greater degree oftransport ofdrug by technetium- labeledOZ-MNEs(i.n.),comparedwithothergroups.Ahmadetal.

reportedthesuccessfulpreparationofanamiloride-NEconsisting of oleicacid, Polysorbate 20andCarbitol1with meandroplet diameter of 89.3611.18nm [48]. From an in vivo study, the AUC0–t in brain following i.n. versus i.v. administration were 7937.46101.19and1254.3725.94ngmin/ml/g,respectively, withasignificantimprovementintheseizurethresholdinrodent modelsofepilepsy.

Oneofthealternativewaystoincreasethepenetrationofthe drugstothebrainthroughthei.n.routecanbetoincreasetheir concentration near the nasal mucus. It is known that mucus containsmucin,acomplexglycoproteincompoundwithsignifi- cant proportions of sialic acid. At physiological pH,sialic acid carriesanetnegativecharge,so,ifthedrug-loadednanocarrierhas a positive zeta potential,through electrostatic interaction, the concentrationandresidencetimeofthedrugneartheabsorption site would beincreased, thereby enhancingthe delivery of the drug intothe brainparenchyma.Jeongandcolleaguesreported thatthetransfectionefficiencyofacationicemulsioncarrierwas

~3-timesbetterthancommonlyusedcommercialliposomecarriers [49]. The increased transfection efficiency using the emulsion carriercouldbeascribedtotheincreasedprobabilityofdelivering intactDNAtothenasalmucosaebecausethecomplexisstrong andinsensitive tomucosaldestabilizers.AcationicNEencapsu- lating ananti-tumornecrosisfactor(TNF)

a

^siRNA(siRNA-NEs), whichhasindicationsinthe preventionofneuroinflammation,

wasdevelopedusingcationiclipidDOTAP[50].Intracellularup- take studies by confocal imaging and flow cytometry showed highercellentryofthedevelopedsiRNA-NEsrelativetoLipofec- tamine¹-transfected siRNA, where siRNA-NEs decreased TNF

a

levelsinlipopolysaccharide(LPS)-stimulatedcellstoasignificant- lyhigherextent.Uponi.n.administrationofsiRNA-NEs,almosta fivefoldhigherlevelwasdetectedintheratbraincomparedwith nonencapsulatedsiRNA.Moreimportantly,i.n.deliveryofsiRNA- NEsdecreasedtheunregulatedlevelsofTNF

a

ⁱⁿ^anLPS-induced modelofneuroinflammation.

Theeffectivenessofcyclosporine-A(CsA)loadedinpositively charged NEs madeof stearylamine (CsA-NEs, zeta potential of +5710mV)wasevaluatedfollowingi.n.andi.v.administration inrats[51].Thebrain-to-bloodexposureratiosof4.49,0.01,0.33 and0.03werefoundforCsA-NE(i.n.),CsA-NE(i.v.),CsA-solution (i.n.)andCsA-solution(i.v.),respectively,indicatingthatCsA-NE hasbeensuccessfulinthedirectnose-to-braintransportofCsA, bypassingtheBBB.Themostlikelyexplanationisthatthepositive chargesonthesurfaceofCsA-NEcanbindstronglytonegatively chargedmucus,thusretainingthedrugatthesiteofabsorptionfor remarkablylongertimes.

Chitosan,apolycationderivedfromnaturallyoccurringchitin, hasattractedagreatdealofinterestforimprovementofthenasal deliveryofpharmaceuticalactivesowingtoitsbioadhesionand permeation-enhancingproperties,and alsodecreasingmucocili- aryclearance, which,collectively,lead toincreased permeation acrossthe nasalmembrane[52].Besides, thepositively charged chitosancouldinteractwiththenegativelychargednasalmucosa [53,54],thus providinga temporaryopening oftight junctions [55]tofurtherhelpthepassageoftheactiveingredientsthrough the nasal mucosa [56]. The in vivo studies revealed that the mucoadhesiveNEofzolmitriptan,preparedbychitosan,provided ahigherAUC0-8andafasteronsetofactionthanthei.v.ornasal solution[57].Inthesameway,Facheletal.reportedusingchit- osan-coatedNEsforrosmarinicacid,withnotablepermeationand retentionofthedruginporcinenasalmucosa[58].Someexamples ofnanoemulsion that havebeen developed forintranasaldrug deliverytothebrainareenlistedinTable2.

Activetargeting

AnotherwaytoenhancetransportofactivesthroughtheBBBisvia receptor-mediatedtranscytosis[59].Applicationsofsomesurfac- tantssuchasP80inthestructureofNEshaveshownpromising resultsincrossingtheBBB.IthasbeenfrequentlyreportedthatP80 canenhancetheadsorptionofapolipoproteinEonthesurfaceof differentnanocarrierswithasubsequent increase inthebrainuptake of P80-coated nanocarriers through the low-density lipoprotein (LDL)receptorsontheBBB.Theparenterallyadministeredcefurox- ime-loadedNE(CFX-NE)exhibitedasignificantCmaxandAUC0–t, prolongedhalf-lifeandreducedclearanceoutofthebrainofrats comparedwiththefreedruginsolution[60].Thisresultcouldbe explainedbytheuseofP80inCFX-NEs,promotingthereceptor- mediatedendocytosisofdrugmoleculesacrosstheBBB.Thisprocess wasnotaconcentration-dependentprocess;hence,ahigherdrug concentrationinplasmawouldnotaffecttherateofbrainuptake.

Usingtheoverexpressedreceptoroftransferrin(Tf)onBBBcells, Prabhakaretal.showedimprovedbraindeliveryofindinavir(IDV) throughtransferrin-coupledNEs (IDV-Tf-NEs)[61].The average

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numberoftransferrinmoleculesattachedtothesurfaceofeachNE dropletwas552.5317.33.Thebraintherapeuticavailabilityof indinavirfromIDV-Tf-NEswas4.69-and1.7-timeshigherthanthe freedrugsolutionandunmodifiedNE,respectively,at6hafteri.v.

injectioninmice,indicatingthepreferentialuptakeofIDV-Tf-NEs byreceptor-mediatedtranscytosis.

L-aminoacidtransporter1isamajorroutefortransportationof branchedor aromatic amino acids throughthe BBB.The entry routeoflipidic

a

^-amino^acids^is^throughcarrier-mediatedendo- cytosis,via bindingtothe receptorandtheninternalizationby endocytosis.Bollamet al.studied theeffect oftwo lipo-amino- acids with different lipid chain length, tetradecyl asparticacid (A14) and decyl glutamic acid (G10), on the concentration of indinavir in brain, where A14-NEs and G10-NEs increased the druglevel~4.27-and2.94-times,respectively,comparedwithfree drugsolution[62].Moreover,incomparisonwiththatofunmodi- fiedNE,thedrugconcentrationinbrainfromG10-NEsandA14- NEsimprovedbetween2.5-and3.38-fold.

LiuandHyedevelopedanintravenousNEofbrain-penetrating peptide K16ApoE (average droplet diameter <200nm) to targetbraintumors[63].K16ApoEpeptideconsistedof16lysine residuesand20aminoacidscorrespondingtotheLDLreceptor bindingsequencefromapolipoproteinEinhealthyandtumor- bearing mice. When 20nmol of K16ApoE peptide NE was injected intravenously, the peptide transiently opened the BBBandallowedevenhigheramountsofNEaccumulationat thebraintumor.

Nanoemulsion application in CNS diseases

Braintumors

Brain tumorsare hard-to-treat neoplasticmanifestations,mostly owingtothehighlylimitedaccessofanticanceragentstothebrain [64].MucoadhesiveNEsloadedbykaempferol(KPF-MNEs)exhib- itedsignificantlyincreasedconcentrationsofthedruginratbrain afteri.n.administration(fivefoldhigherthanthefreedrug)[65].In addition,KPF-MNEsdecreasedC6gliomacellviabilityviainduction ofapoptosistoasuperiordegreeascomparedwiththefreedrug.The cytotoxicityofKPF-MNEsontheC6gliomacelllinewas~20-fold betterthanthefreedrugbecause ofthehigherlevelofcellularuptake whenthedrugwasincorporatedintomucoadhesiveNEs.Castilho- Fernandesetal.showedthatchloroaluminumphthalocyanineNEs (AlClPc-NEs)inducecelldeathinU87-MGcellswithadose-depen- dentmechanismand,possibly,couldserveasanappropriateadju- vanttreatmentformalignantglioma[66].Theirstudyrevealedthat caspase-9activitywashigherthanthatofcaspase-3;so,AlClPc-NEs promptedapoptosisinU87-MGcellsmostlythroughthemitochon- drialintrinsicroute.Inthisway,a fairlylowdose offisetinNEs reduced~53%oftumorvolume inLewislung-carcinoma-bearing mice,whereasasixfoldhigherdosewasrequiredtoobtainasimilar tumorgrowthinhibitioninthecaseoffreefisetin[67].Meanwhile, theimprovedbioavailabilityofthefisetinNEsispossiblyduetothe uniquelymphaticdistributionfollowingintraperitoneal(i.p.)administration,whichcanbeconsideredasafavorablecharacteristic, particularlyinthecaseofanticanceractivesthatmustreachlymph nodesinmetastases.

TABLE2

Nanoemulsionsemployedinintranasaldrugdelivery

Drug Composition Method Size(nm) Outcomes Refs

Paroxetine Capmul¹MCM/Solutol¹HS 15/Propyleneglycol

58.473.02 A2.57-foldenhancementinpermeationas comparedtotheparoxetinesuspension Effectiveinenhancingthedepressedlevelsof glutathioneanddecreasingtheelevatedlevelsof TBARS

[102]

Paliperidone Labrafil¹/M1944CS/

Polysorbate80/Tween120

38.25 Significantdifferenceinthelocomotoractivity whencomparedwithplaindrug

[103]

Resveratrol Curcumin

Hyaluronicacid Spontaneous

emulsification

115.20.15 Managedtoincreasetheamountsofthetwo polyphenolsinthebrain(about7-and9-fold increaseinAUC0–7hforresveratrolandcurcumin, respectively)

[104]

Tramadol Isopropylmyristate/

Tween120/Labrasol¹

Magneticstirring 136.34.3 Displayedsignificantlyhigherantinociceptive effectandAUC_0!24hascomparedwithtramadol solution

[105]

Pretomanid Octadecylamine/Tween1 80/Asolectin

Sonication 186.460.38 Peakconcentrations(Cmax)inbrainof12062.3ng/

gthatissignificantlyhigherthantherequired therapeuticlevel

[106]

Selegiline Grape-seedoil/Solutol¹/ Labrasol¹

Highenergy emulsification

61.434.10 Significantimprovementinbehavioralactivitiesin Parkinson’sdiseaseinrats

[107]

Resveratrol VitaminE/Sefsol¹/ Polysorbate80/Transcutol¹ P

Spontaneous emulsification,High- pressure

homogenization

1021.46 ThelevelsofGSHandSODweresignificantly higherandthelevelofMDAwassignificantly lowerintheresveratrol-nanoemulsion-treated Parkinson’sdiseasegroup

[108]

Safranal Tween120/Labrasol¹/ Cremophore¹EL/Carbitol¹

Titrationmethod 89.649.12 Inducedcerebralischemiaratsexhibited significantimprovementinneurobehavioral (locomotorandgripstrength)andantioxidant activity

[48]

Zaleplon Cremophor¹/RH40/

Transcutol¹HP/Miglyol¹

44.573.4 Significant3-foldincreaseinplasmaandbrain GABAlevelscomparedwiththemarketedproduct sleepaid

[109]

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Theuse ofClinOleic,acommerciallyavailableparenteralnu- trition NE for the delivery of paclitaxel (PX), was reported by Najlahetal.[68].PX-loadedClinOleicdecreasedtheviabilityto 34.57%forSVG-P12cells(normalglialcelllines)andto6.4%for U87-MGcells(gradeIVgliomacelllines).Meanwhile,PX-loaded ClinOleicalsoshowedasignificantlylower(P<0.05)cytotoxicity towardSVG-P12thanthatoffreedrug,indicatingabetterselec- tivity againstthemalignantcells.Cancerous cellsareknownto dividefasterthannormalcells;hencetheirintakeofnutrientsis faster;thismightexplainthereasonbehindtheselectivityofthe PX-loadedClinOleictowardthecancerouscells–thepresenceof oliveoil and/orsodium oleateinClinOleic compositionmight, additionally,haveafurtherenhancementeffectonthetargeting propertiesofthisformulationtowardtheU87-MGcells.

SimultaneousstudiesonNEandsolutionformsofananticancer substanceisolated fromthe traditionalChineseherb magnolia:

honokiol(HK),afteri.v.administrationtotumor-burdenedmice was carried out [69]. The NEs with a mean diameter of 186.61.7nm were prepared from soybean lecithin, Synperonic1 F68 and soybean oil. The half-life and targeting propertiestotumortissuesofHK-NEswereremarkablyimproved compared with those seen with the free drug solution. in vivo studiesindicatedthatHK-NEtreatmentcausedsignificantinhibi- tionof mousesarcoma S180tumor growth comparedwith the solutionofHK.Inasimilarstudy,DHA-SBT-1214wasincorporated inafish-oil-based NE,withanaveragediameterof200nm [70].

TheNEformulationdisplayedanimprovedPKprofilealongwith higherexposureofthedrugtothetumorandalmostdoubledthe tumor-targetingefficiencycomparedwiththefreedrugsolution.It wasreportedthattheIC₅₀valuedecreasedwhenthebraincancer cellsweretreated witha flaxseed-oil-NEformulationofchemo- therapeuticagents,modifiedbya

m

-opioid-receptor-targetingli- gand(USpatentno.WO2016090092A1).

Neurodegenerativedisorders

Neurodegenerative disorders encompass a range of conditions characterizedby thepersistent andprogressivelossofneuronal subtypes. One of the most commonforms of dementia in the elderlyisAlzheimer’sdisease,affecting>35millionpeopleworld- wide,andthenumberofpeoplewithAlzheimer’sdiseaseincreases annually [71]. Similar to other CNS diseases, the BBB is the formidableobstaclefordrugsaccessingthebrainandsomeaspects oftheBBBinAlzheimer’sdiseasepatientscanevendifferfromthe normalBBB,suchasP-gpfunction,cerebralbloodflowandCSF reabsorption.

Inaninterestingstudy,followingoridonin-NE(OD-NE)injec- tion,numbersofamyloidplaquesinthecortexofAPP/PS1miceas wellasareapercentagesof

b

^-amyloid^(A

b

⁾^deposition^were^signif-

icantlyreducedcomparedwith thecontrolgroup[72].Further- more,the impairednestconstructionbehaviorwassignificantly restoredbyOD-NEtreatment.Itwasreportedthattheenhanced drugavailabilityinbrain,afteri.n.administrationofrivastigmine NEs (RVN-NEs)comparedwithfreedrugsolution,wasprobably the result of small globule size, high drug release and longer residence time of the RVN-NEs in the nasal cavity [73].Small globulesizepotentiallyallowsRVN-NEstobemovedtranscellu- larlythrougholfactoryneuronstothebrainbytheseveralendo- cytic trails of sustentacular or neuronal cells in the olfactory

membrane. In addition, use of P80 and Transcutol¹ P in the NEs significantly improved the nasal absorption of the drug, possiblyasaresultofP-gpinhibition.

Thymoquinone-richfraction NEs (TQRF-NEs) reducedsoluble A

b

⁴⁰^and^A

b

^42,^partially^via^modulation^of^A

b

^precursor^protein

processingwhichstops:A

b

generation;theupregulationofinsulin- degradingenzyme,anenzymethatdegradesA

b

^;^theupregulationof LRP1,aproteinthattransportsA

b

brain-to-blood;thedownregula- tionofBACE1,anenzymethatinitiatesA

b

production;andthe downregulationofRAGE,areceptorthattransportsA

b

^from^blood

intothebrain[74].Thus,TQRF-NEsemployseveralmechanismsfor decreasingA

b

accumulationinthebrain,byinducingreductionof A

b

^generationâs^wellâsîncreasingîtsdegradationandclearance fromthebrain.Ismailetal.reportedthattheprotectiveeffectsof TQRF-NEsagainstahigh-fat-cholesterol-diet-inducedhypercholes- terolemiawererelatedtoareductionincholesterolandA

b

^levels,

and growthof antioxidantlevels [75].The cholesterol- and fat- enricheddietusedinthatstudyexhibitedpro-oxidantactivitythat couldtriggerAlzheimer’sdiseasebiomarkerssuchasA

b

^.^Therefore,

TQRF-NEs,possiblythroughtheirantioxidantproperties,wereable toshieldagainstthisearlierdestructivedamagetowardAlzheimer’s diseasepathology.

Selegiline NEs (SG-NEs) were developedfrom grape-seed oil, Sefsol218¹,P80andLauroglycol¹90byhigh-pressurehomoge- nization[76].TheSG-NEsshoweddropletsizeandzetapotentialof 61.434.10nm and 34mV, respectively. Biochemical evalua- tionrevealedanincreaseinthe extentofantioxidantenzymes, whereas the thiobarbituric-acid-reactive substances were de- creasedini.n.SG-NE-treatedanimalsascomparedwithhaloperi- dol-induced Parkinson’sdiseaserats (control).Additionally,the brain dopamine concentrations of SG-NE-treated and control groupswere16.613.06and 8.591.00ng/ml,respectively. In conclusion,SG-NEsweresuccessfulinreducingthedopamineloss, whichsuggeststheycouldbeapotentialtechniqueforselegiline deliverytoreducethedamagecausedbyfreeradicals,aswellas avoiding subsequent biochemical changes that arise during Parkinson’s disease. Similarly, the increase in the content of glutathione and decrease in the extent of lipid peroxidation showed the beneficial effectsof the transdermal ropinirole NE inParkinsonismrats[77].Thedepletedlevelwasrestoredsignifi- cantly(35.09%)intheNE-receivinggroupincontrasttothefree drugsuspensiongroup,wheretherestorationratewasonly13.3%.

Amyotrophiclateralsclerosis(ALS),amotorneurondisease,isa progressiveneurodegenerativedisordercharacterizedbythedete- riorationofupper andlowermotorneurons.Todate,onlyone drug(i.e.,riluzole)hasbeenapprovedforthetreatmentofALS.

ParikhandPateldevelopedriluzole-loadedNEs(RZ-NEs)byphase titrationfromSefsol¹218,P80andCarbitol¹,withadropletsize of23.920.52nm[78].Brainuptakeofriluzoleposti.n.administration of RZ-NEs was significantly higher compared with oral administrationofRZ-NEs.

HIV-associatedCNSdisorders

TheabnormalimmuneresponsescausedbyHIVinfectioncanleadto neurological illnesses[79].It isveryimportantfor the treatment regimensofHIV-associatedneurologicalproblemstobeabletopass throughtheBBBwithaproperconcentration.Itiswell-knownthat thebrainpenetrationofthemajorityofanti-HIVdrugsislimited

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mainlybecausethesecompoundsarethetypicalsubstratesoftheABC transporterslocatedattheBBBandBCSFB[80].

The developed saquinavir mesylate NEs (SQVM-NEs) from Capmul¹MCM,P80andPEG400enhanced thedrugpermeation intothebraincomparedwiththefreedrugsuspension[81].Gamma scintigraphyimagingconfirmedthetransportofSQVMintoratbrain atgreaterlevelsafteri.n.administrationofSQVM-NEs.Moreover,in PK studies, the brain level of indinavir subsequent to administration of P80-containingNEwassignificantly(P<0.05)higherthanthatpro- ducedbytheadministrationofafreedrugsolution(2.44-fold)ora P80-lackingindinavir-loadedNE(1.48-fold)[82].Theincreasedbrain- specificaccumulationofindinavirbymodifiedNEispossiblyattrib- utabletotheenhancedLDL-mediatedendocytosisas wellasP-gp inhibitionbyP80attheBBB.Treatmentoftoxoplasmosisisregularly requiredinimmune-compromisedpatients.AtovaquoneNEs(AQ- NEs) were prepared from grape-seed oil by spontaneous emulsification [83].Afteroraladministration,AQ-NEsshowedimprovedoralbio- availabilityandsurvivaltimeinmicealongwithdecreasedparasite- miaandnumberandsizeofbraincysts.

Ischemicstroke

Ischemicbrainstrokeisoneofthechallengingcausesofdeathand disabilityinmajorindustrializedcountries.Thefatalityofstrokeis becauseofanarrowtimewindowfortherapeutictreatment,afast progressingpathology(~3h)andlackofviablestrategiestoovercome thediseasepathology[84].ThymoquinonemucoadhesiveNEs(TQ- MNEs) showed remarkable brain-targeting efficiency (628.578644.79%)andbrain-targetingindex(89.972.94%)fol- lowingi.n.administration[85].Improvedneurobehavioralfunction (locomotor andgripstrength) was discoveredin middle-cerebral- artery-occlusion-inducedcerebralischemicratsfollowingtheadmin- istrationofTQ-MNEs.AstudybyGorainetal.showedsignificantly higher brain concentrations of olmesartan (0.2900.089mg/ml, 0.3330.071mg/mland0.2170.062mg/mlat0.5,2.0and8.0h postdosing,respectively)whenadministeredorallyasaNEformula- tion,comparedwiththeaqueousdrugsuspension[86].

Quercetinisapolyphenolwithanantioxidanteffectinvitro, butithaslimited bioavailabilityin vivoowingto itshighlipo- philicity. Quercetinmucoadhesive NEs(Qu-MNEs)were developed through ionic gelation from oleic acid, PEG 400, P80, Labrasol¹ and Transcutol¹ HP with mean droplet size of 91.634.36nm[87].Someimportantfindingssuchasimproved neurobehavioralfunction(locomotor andgripstrength),histo- pathologyandreducedinfarctionvolumeeffectsweredetectedin middle-cerebral-artery-occlusion-inducedcerebral ischemic rats afteri.n.administrationofQu-MNEs.Galhoetal.evaluatedthe effectofaquercetin-loadedNE(Qu-NE)preparedfromcastoroil, lecithinandPEG-660stearatewithameandropletsizeof~20nm in a collagenase-induced intracerebral hemorrhage rat model [88].AnimalstreatedwithQu-NEsshowedasignificantimprove- mentinthebeam-walkingandopen-fieldtests.Similarly,Qu-NEs

decreasedthesizeofthehematoma,whilepreservingtheactivity of glutathione-S-transferase, with increased glutathione (GSH) level,andthetotalantioxidantcapacity.Theauthorsconcluded that the administration of the NE formulation of quercetin increased itsantioxidanteffect,which wasreflectedin theim- provementofmotorskillsaswellasthehematomasizedecrease.

Schizophrenia

Schizophrenia isadisablingpsychiatricdisorderaffectingmany peopleworldwide.Itmanifestsinavarietyofsymptomsranging frommisinterpretationofrealityandillusionstodisorganization ofthinkingandbehavior.Itisassociatedwithprogressivealtered brainfunctionsduringthecourseoftheillness[89,90].Quetiapine (QTP),anantipsychoticdrug,getsextensivelymetabolizedbythe liver.ToincreasebioavailabilityofQTPviabypassingitshepatic first-pass effect,aQTP-loadedNE(QTP-NE)wasdevelopedfrom Capmul¹MCM,P80,Transcutol¹Pandpropyleneglycol,then administeredi.n. in Wistarrats[91]. QTP concentrationin the brainuponadministrationoftheNEwasobservedtobehigherat alltimepointsthanfreedrugsolution,indicatingtheefficiencyof braintargetingofQTP-NEsafteri.n.delivery.

invivoexperimentsofrisperidoneNEsstabilizedwithP80(RSP- NEs)revealed1.2–1.5-foldenhancedrelativebioavailability,1.1–

1.8-fold reduced liver accumulation and nearly 1.3-fold higher brainentryofdrugposti.p.administrationofRSP-NEscompared with free drug solution in a rat model[92].Moreover, from a behavioralstudy,RSP-NEsexhibitedadecreaseinbasalandam- phetamine-inducedlocomotoractivityin rats.RSP-NE-receiving animalsalsoshowedanearlyonsetofantipsychoticactionwhich lastedfor~90minpostinjection.

Concluding remarks

Thepresentreviewhasattemptedtodemonstratethepotentialof NEs, inthefieldofdrug deliveryto thebrain,asefficienttools challengingthelimitedbrainentryofawidevarietyoftherapeutic agents.NEs arekinetically stable dispersionsoftwo immiscible liquidswithinterventionofemulsifier(s)andenergy,withamean dropletdiameterof20to500nm.Theiruniquestructuresaffect thesolubilityandstabilityoftheincorporateddrugs,alongwith themodulationoftheirinteractionwiththebrainendothelialcell membrane.Itiswell-documentedby severalinvivostudiesthat NEs can be considered as one of the most promising delivery systems for the CNS-active agents used as partof the efficient regimensagainstthehard-to-treatCNSdiseaseslikebraintumors, AIDS-related dementia,Alzheimer’sdisease,Parkinson’sdisease, strokeandschizophrenia.However,owingtothedifferentaspects tobeconsideredseriouslybeforethewidespreadclinicalapplica- tion of this delivery system, extensivestudies are still needed, especiallyinthefieldoftoxicity,costandefficacyoftheprotocols forthisnoveldrugdeliverysystemintheclinicalsetting.

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