Engineering5(2019)69–75Contents lists available at ScienceDirectEngineeringResearch
TraditionalChineseMedicine—Review
AndrographolideLoadedinMicro-andNano-Formulations:
ImprovedBioavailability,Target-TissueDistribution,andEf?cacyofthe‘‘KingofBitters”
MartaCasamonti#,LauraRisaliti#,GiuliaVanti,VieriPiazzini,MariaCamillaBergonzi,AnnaRitaBilia?DepartmentofChemistry‘‘UgoSchiff”,UniversityofFlorence,SestoFiorentino50019,Italyarticleinfoabstract
Andrographolide (AG) is the characteristic constituent of Andrographis paniculata, of the Acanthaceae family. This plant is a well-known Asian medicinal plant that is widely used in India, China, and Thailand. A monograph of Herba Andrographidis (Chuanxinlian) is included in the Chinese Pharmacopoeia, which reports that this decoction can ‘‘remove heat, counteract toxicity, and reduce swellings.” The numerous potential activities of AG range from anti-in?ammatory to anti-diabetic action, from neuroprotection to antitumor activity, and from hepatoprotective to anti-obesity properties. However, AG has low bioavailability and poor water solubility, which can limit its distribution and accumulation in the body after administration. In addition, AG is not stable in gastrointestinal alkaline and acidic environments, and has been reported to have a very short half-life. Among the diverse strategies that have been adopted to increase AG water solubility and permeability, the technological approach is the most useful way to develop appropriate delivery systems. This review reports on published studies related to microparticles (MPs) and nanoparticles (NPs) loaded with AG. MPs based on polylactic-glycolic acid (PLGA), alginic acid, and glucan derivatives have been developed for parenteral oral and pulmonary administration, respectively. NPs include vesicles (both liposomes and niosomes); polymeric NPs (based on polyvinyl alcohol, polymerized phenylboronic acid, PLGA, human serum albumin, poly ethylcyanoacrylate, and polymeric micelles); solid lipid NPs; microemulsions and nanoemulsions; gold NPs; nanocrystals; and nanosuspensions. Improved bioavailability, target-tissue distribution, and ef?cacy of AG loaded in the described drug delivery systems have been reported.
ó 2019 THE AUTHORS. Published by Elsevier LTD on behalf of Chinese Academy of Engineering and Higher Education Press Limited Company. This is an open access article under the CC BY-NC-ND license
Articlehistory:Received6September2018Revised25October2018Accepted12December2018Availableonline04January2019Keywords:AndrographispaniculataAcanthaceaeAndrographolideDeliverysystemsMicroparticlesandnanoparticleImprovedbioavailabilityTarget-tissuedistribution1.IntroductionAndrographispaniculata(Burm.f.)Neesisawell-knownAsianmedicinalplantoftheAcanthaceaefamily(Fig.1).ItiscalledFa-Tha-Lai-JoneinThailand[1];KalmeghinAyurvedicmedicine;andChirettaor‘‘kingofbitters”inIndia,China,andThailand[2,3].AmonographofHerbaAndrographidis(Chuanxinlian)isincludedintheChinesePharmacopoeia,whichreportsthatthisdecoctionactsto‘‘removeheat,counteracttoxicity,andreduceswellings”[4].Traditionalindicationsfortheuseofthisdecoctionincludeeffectsintheupperrespiratorytract,suchasthecommoncold,bronchitis,sinusitis,pharyngotonsillitis,whoopingcough,?Correspondingauthor.#E-mailaddress:ar.bilia@uni?.it(A.R.Bilia).Theseauthorscontributedequallytothismanuscript.
pneumonia,andotitismedia.Inaddition,activityagainstdiarrhea,enteritis,lowerurinaryinfections,dermatitis,andtuberculosishastraditionallybeenreported[4–6].Activityagainstthecommoncold,pharyngotonsillitis,uncom-plicatedsinusitis,bronchitis,acutediarrhea,andurinaryinfectionsisalsosupportedbyclinicaldatareportedintheWorldHealthOrganization(WHO)monographonHerbaAndrographidis[7].ThemainactiveconstituentsofHerbaAndrographidisarebicyclicditerpeneswithac-lactonemoiety,whichprincipallyincludeandrographolide(AG,Fig.2)anditsanalogs,14-deoxyandrographolide,neoandrographolide,and14-deoxy-11-12-didehydroandrographolide[8].InadditiontothenumerouspotentialactivitiesofAG,whichrangefromanti-in?ammatorytoanti-diabeticaction,fromneuroprotectiontoantitumoractivity,andfromhepatoprotectivetoanti-obesityproperties[9],AGhaspoorwatersolubility,whichlimitsitsdistributionand70M.Casamontietal./Engineering5(2019)69–75Fig.1.Andrographispaniculata(Burm.f.)Nees,Acanthaceae.Fig.2.Andrographolide(AG),themaincharacteristicditerpenoidinAndrographispaniculata.accumulationinthebodyafteradministration.Inaddition,AGisunstableingastrointestinalmediaandpossessesaverylimitedhalf-life(circa(ca.)2h)[10].2.MicroparticlesandnanoparticlesloadedwithAGAGisaverypromisingnaturalproductwithvariouspotentialtherapeuticbene?ts;however,ithasnotreacheditsmilestonetherapeuticpotentialduetoitslowbioavailabilitywhenadminis-teredinconventionaldosageforms.ThedevelopmentofsuitabledeliverysystemsforAGisanurgentissueinthedevelopmentofef?cacioustherapeuticapproachesforthiscompound.AmongthediversestrategiesthathavebeenadoptedtoincreaseAGwatersolubilityandpermeability,thetechnologicalapproachisthemostusefulwaytodevelopsuitabledeliverysystems.Averysimpleapproachtoimprovethesolubilityofamoleculeistomodifytheparticlesizethroughmicronizationandnanonizationtechniques,whichcanenhancethetotalsurfaceareawithaconsequentimprovementofthedissolutionbehavior.Microparticles(MPs)—solidsorsmalldropletsofliquidsenclosedinnaturalorsyntheticpolymersofvariablethicknessandpermeability—arefrequentlyusedinthisendeavorinordertoobtainacontrolledreleaseoftheencapsulateddrug.Bothorganicandinorganicnanoplatforms(Fig.3)canalsoincreasesolubilitytoanextraordinarydegree,andcanenhancephotostability,chemicalstability,bioavailability,andtissuedistribution.Thesenanocarrierscanovercomemultidrug-resistancephenomenaandcrossbiologicalbarriers,includingtheblood–brainbarrier(BBB).Organicnanoparticles(NPs)includepolymericandlipidNPs.AmonglipidNPs,vesicles,microemulsions(MEs),nanoemulsions(NEs),andsolidlipidNPs(SLNs)arethemostwidelyinvestigated.Finally,thesimpleuseofnanopowdersranginginsizefrom10to1000nmareanalternativewaytoenhancethesolubilityofAG,principallyduetotheincreaseddissolutionpropertiesthatresultfromthegreatersurfaceareasincomparisonwithsimilarmassesoflargerscalematerials.Fortheseformulations,whicharemainlysuitablefororalorparenteraladministration,surfactantsorhydrophilicpolymersshouldbeaddedduringmillingforsurfacestabilization,inordertoinhibittheformationofaggregates.2.1.MicroparticlesMPsincludemicrospheresandmicrocapsules.Amicrocapsuleischaracterizedbyaninternalcoreandanexternalshell.Theinternalcorecanbeliquid(i.e.,oilorwater)orsolid,andtypicallycontainstheactiveingredient,whiletheshellisusuallyapolymerorwax.Amicrosphereisasolidmatrixparticle;theactiveingredientisusuallydissolvedormeltedinthematrix.Astudyinvestigatedthedevelopmentofsustained-releasemicrospheresbasedonthecopolymerobtainedfromlacticandglycolicacid—polylactic-glycolicacid(PLGA)—loadedwithAG.PLGAmicrosphereswerepreparedbyanemulsionsolventevaporationmethod.Theformulationwasoptimizedusingresponsesurfacemethodology,whichwasusedtoidentifytheoptimumlevelsoftheprocessvariables,whichhadsigni?canteffectsonparticlesizeandentrapmentef?ciency.Thestudyfocusedonthedevelopmentofsphericalmicrosphereswithanaverageparticlesizeof(53.18±2.11)lm.Theentrapmentef?ciency(EE)was75.79%±3.02%,whilethedrugloadingwas47.06%±2.18%.ThereleasekineticsfollowedtheKorsmeyer-Peppasmodel,withalowinitialburstfollowedbyasuccessivepro-longedrelease(upto9d).AfterintramuscularadministrationoftheMPs,AGplasmaconcentrationswerefoundtobemoderatelyhighoveraperiodofoneweek[11].ItisgenerallyacceptedthathighdosesofAGprovidehepaticprotection;however,duetotheextremelybitterpropertiesofAG,sicknessandnauseacanfrequentlyoccur.Toaddressthisissue,bitterlessMPspreparedwithsodiumalginateandcalciumionsweredevelopedusingvariousAG:alginateratios(i.e.,1:2,1:1,and2:1).ThereleasekineticsofthedevelopedMPs?ttedwellwiththeKorsmeyer-Peppasequation:TheMPsreleasedatacidicpH(1.2or4.0)withca.15%releaseforupto4h;acompletereleaseoftheremainingAGthenoccurredatpH7.4.ThedevelopedMPswereveryusefulfortheoraldeliveryofAGandhadagoodEE,with86%release[12].Inafurtherstudy,thesameauthorsinvestigatedthemechanismofgelationfortheentrapmentofAG.AGstabilitywasobtainedinthecross-linkedMPs[13].Amicronizedformula-tionforadrypowderinhaler(DPI)wasalsodevelopedusinganat-uralpolysaccharide,scleroglucan.TheMPsdisplayedameanaerodynamicdiameterof(3.37±0.47)mm.TheinvivostudiesexhibitedasuitableAGlungdeposition,andnoin?ammationortoxicitywasfoundafter24h.Moreover,theformulationdemon-stratedimprovedactivityinpulmonaryarterialhypertension[14].2.2.Nanoparticles2.2.1.PolymericnanoparticlesPolymericnanoparticles(PNPs,Fig.3)arethemostwidespreaddeliverysystems.Theseincludenanospheresandnanocapsules.Nanospheresarematrixsystemsinwhichthedrugishomoge-neouslydistributed,whileinnanocapsules,thedrugiscontainedinacore,whichiswalledinbyanoutershell.PolymersusedforthepreparationofPNPsareclassi?edassyntheticornatural(i.e.,M.Casamontietal./Engineering5(2019)69–7571Fig.3.Nanocarriershavebeendevelopedtoimprovethebioavailability,target-tissuedistribution,andef?cacyofAG,the‘‘kingofbitters.”NPs:nanoparticles;PNPs:polymericnanoparticles;SLNs:solidlipidNPs;MEs:microemulsions;NEs:nanoemulsions.biopolymers),andarecommonlyclassi?edasbiodegradableornon-biodegradableaswell.Biodegradationofapolymerinvolvesthecleavageofhydrolyticorenzymaticbondsinthepolymer,leadingtopolymererosion.Mostnaturallyoccurringpolymersundergoenzymaticdegradationandarethusthe?rstbiodegradablebiomaterialsusedinpharmaceuticaltechnology.Naturalpolymersincludeproteins(e.g.,albumin,gelatin,soyproteinhydrolysate,andcasein)andpolysaccharides(pectin,cellulose,starch,gumarabic,carrageenan,alginate,xanthangum,gellangum,andchitosan)[15].Royetal.[16]developedPLGAPNPsloadedwithAGandcoatedwithchitosanforantitumortherapy.ThePNPswerenontoxicandincreasedtheanticanceractivitybythreefoldintheMCF-7celllineandinvivostudiesusingEhrlichascitescarcinoma(EAC)cells,incomparisonwithunformulatedAG.TheanticanceractivityofAGloadedinotherPNPswastestedforspeci?ctargets.Kimetal.[17]formulatedwater-solublePNPsforsystemicadministration,usingapolymerobtainedfromphenylboronicacid.Thisnanocar-rierdisplayedexceptionaltargetingpropertiesbothinvitroandinvivo,andresultedinasigni?cantdecreaseofinvivotumorgrowth.Royetal.[18]preparedAGloadedinPLGAPNPs(50:50),whichwerestabilizedwithpolyvinylalcohol(PVA).ThestudyfocusedonAGdeliveryintomacrophagecellsinfectedwithleish-manialparasite.ThePNPaveragesizewas173nm,withasurfacechargeofà34.8mV.Activityontheinfestedmacrophageswassig-ni?cantinthecaseofPNPscontaining4%PVA(IC50of34lmoláLà1),whichisaone-quarterdoseofpureAG(IC50of160lmoláLà1).ThesameauthorsrecentlypreparedPNPswithhepatoprotectiveactiv-ityforuseinhepatotoxicconditions.Cationicmodi?edPLGAPNPsloadedwithAGweredeveloped,andshowedsuperiordissolutionbehaviorincomparisonwithpureAGandfavorablecytokineregu-lationinthehepatictissues,leadingtoarapidrecoveryofmouseliver[19].Moreover,in2017,Dasetal.[20]exploredtheeffective-nessofAGencapsulatedinPLGAPNPsagainstliverdamageinmiceinducedbyarsenic.ThePNPshadanaveragediameterof65.8nmandtheEEwas64%.TheNPsincreasedthelevelofreducedglutathioneandantioxidantenzymes,includingsuperoxidedismutaseandcatalase.Theprotectiveef?ciencyofAGloadedinPNPswasabout?vetimesgreaterincomparisonwithunformu-latedAG.NPadministrationimprovedthelivertissuearchitecture,suggestingabene?cialeffectagainstarsenic-inducedlivertoxicity.AGhasalsobeenreportedtoameliorateneurodegenerativedisor-ders.Toovercomeitslowbraindistribution,Guccioneetal.[10]loadedAGintoalbuminNPs(ANPs)andethylcyanoacrylateNPs(ENPs).TheabilityofbothNPstopermeatetheBBBwasinvesti-gatedusinganinvitroBBBmodel,hCMEC/D3.AlthoughAGwasunabletocrosstheBBBmodeltoanysigni?cantdegree,theANPsimprovedthepermeationofAGbytwofold.Inaddition,theinteg-rityofthecelllayerwasmaintained.Incontrast,whileENPsincreasedthecrosspropertiesofAG,theBBBwastemporarilydis-ordered.AnothernanovectorthatwastestedinvolvedpH-sensitivePNPsbasedonacationicpolymethacrylatecopolymer(EudragitòEPO).TheoptimizedformulationconsistedofPluronicòF-68(0.6%,w/v)andEudragitòEPO(0.45%,w/v).IthadaveryhighEE(93.8%±0.67%),homogeneousparticlesize((255±9)nm),andgoodsuper?cialchargevalues((29.3±3.4)mV).Theinvivoabsorp-tionofAGandofAGloadedintheNPswasstudiedatadoseof10mgákgà1inmaleWistaralbinorats.TheAGloadedinPNPscausedatremendousincreaseinareaunderthecurve(AUC0–1)(ca.2.2fold)andthemaximumconcentration(Cmax)(3.2fold)incomparisonwithunformulatedAG.Inaddition,therelativebioavailabilityincreasedby121.53%(P<0.05)incomparisonwithpureAG.OtherparameterswerefavorablyaffectedbytheloadingofAGinNPs:AsmalleramountoftimethatAGispresentatthemaximumconcentrationinserum(Tmax)(4.0fold)andareductioninCl/F(2.2fold)wereobserved[21].2.2.2.PolymericmicellesPolymericmicelles(Fig.3)areself-assemblingcolloidsofamphiphilicpolymers,whichcanspontaneouslyaggregateinaparticularsolvent(generallywater).AGwasentrappedinamicellarformulationbasedonanamphiphilictriblockcopolymerofD,L-lacticacid,glycolicacid,andethyleneglycol(poly(lactide-co-glycolide)-block-poly(ethyleneglycol)-block-poly(lactide-co-glycolide)(PLGA-PEG-PLGA)),andthenevaluatedforbioavailabilityinvivoandforinvitrocytotoxicity.Cellularuptakeand72M.Casamontietal./Engineering5(2019)69–75cytotoxicity,includingcellcyclearrest,proliferationinhibition,andpro-apoptosiseffectsweretestedagainsthumanbreastcancerMAD-MD-231cells.Theloadingef?ciencyofthemicelleswasabout92%,andtheparticlesizewas(124.3±6.4)nm.ThemicellesexhibitedahigherinhibitionofproliferationwhencomparedwiththeunformulatedAG.Thehighesteffectivenessofcellularuptakeandintracellulartransport,pro-apoptoticproperties,andcellcyclearrestattheG2/MphasewerefoundinMAD-MD-231cellsusingthemicelles.Pharmacokineticsstudieswerecarriedoutinrats,andboththemeanresidencetimeandplasmaAUC0–1werefoundtoincreasebyalmostthreefoldincomparisonwithunformulatedAG[22].Inafurtherstudy,aseriesofcopolymersobtainedfrommethoxypoly(ethyleneglycol)-poly(D,L-lacticacid)(mPEG-PLA)withvariousratiosofhydrophilictohydrophobicportionswassynthetizedtoencapsulateAG.Themicelleshadasizeof(92.84±5.63)nm,ahighEEof91.00%±11.53%,andaloadingcapacityof32.14%±3.02%(w/w).mPEG-PLAloadedwithAGwasfoundtohavegoodstabilityagainstsaltdissociation,proteinadsorption,andanionsubstitution.ThesolubilityofAGandaderivativeofAG(14-deoxy-11,12-didehydroandrographolide)inmicellesincreasedby4.51timesand2.12timesinwaterincomparisonwithunformulatedAG.mPEG-PLAloadedwithAGshowedthesamereleasepro?leinadifferentdissolutionmedium.CytotoxicitytestinginvitrodemonstratedthatAGloadedinmPEG-PLAexhibitedhighercellviabilityinhibitioninmousebreastcancer4T1thanfreeAG[23].2.2.3.VesiclesVesicles(Fig.3)arecolloidalvectorsformedbybilayers,whichcanloadhydrophilicandhydrophobiccompounds.Liposomesareprincipallyconstitutedofnaturalphospholipidsandcholesterol,whileniosomesarenonionicsurfactant-basedvesicles[24].AveryrecentstudybyKangetal.[25]reportedonliposomalco-deliveryofAGanddoxorubicintoinhibitbreastcancergrowthandmetastasis.Theliposomewaspreparedwithacell-penetratingpeptide,whichwasabletoinhibittheinvitroproliferationof4T1cells.Twodistinctivetestsweredone:awound-healingassayandatranswellinvasionassay.TheliposomewasfoundtoenhanceAGaccumulationintumorsandtoresultinhighintra-tumorpenetrationinatumormousemodelofbreast.AsynergisticeffectofdoxorubicinandAGwasfound.Inanotherstudy,AG-loadedniosomeswereprepared.TheniosomesimprovedthetissuedistributionandbioavailabilityofAGinmice.Inparticular,AG-loadedniosomesaccumulatedinthelivermuchmorethanthefreedrug.Invitrostudiesonanti-hepatocellularcarcinoma(HCC)ef?cacyinHepG2cellsdisclosednosigni?cantdifferencesbetweenthefreedrugandAG-loadedniosomes[26].Inafurtherstudy,loadingAGinanaturalsoya-phosphatidylcholinemixturewasfoundtoenhancetheabsorptionandhepatoprotectiveactivityofAGincomparisonwithunformulatedAG.ThestudyprovedthehepatoprotectivepotentialofAGusingaratmodelofhepatotoxicityinducedbycarbontetrachloride.Theresultsshowedsigni?cantlyincreasedabsorption,bioavailability,andhepatoprotectivepotentialofAGloadedinvesicleswhencomparedwiththeunformulateddrug.TheeffectsofAG-loadedvesicleswerecomparabletothoseofsilymarin,whichisusedasastandarddrug[27].Maitietal.[28]obtainedsimilarresultsinastudyusingthesameformulationinrats.TheformulationhadanimprovedbioavailabilityandabetterhepatoprotectiveactivitythanunformulatedAG.Theformulationwasveryhelpfulinsolvingtheproblemofrapidclearanceandloweliminationcausedbytheshorthalf-lifeofAG.Inanadditionalstudy,Sinhaetal.[29]reportedthecapacityofAGencapsulatedinliposomeanddecoratedwithmannosylorfucosyl(asactivetargetingformacro-phages)toreducehepaticandrenaltoxicity.Furthermore,adecreasewasobservedintheparasiticburdenofexperimentalleishmaniosisinahamstermodelandinthesplenictissuehistologicalarchitecturewhencomparedwithtreatmentwithunformulatedAGorconventionalAG-loadedliposomes.Inafurtherstudy,Lietal.[30]developedAG-loadedliposomaldrypowderinhalers(LDPIs)forpulmonarydeliveryforthetreatmentofpneumoniainducedbyStaphylococcus(S.)aureus.TheAG-loadedliposomeswerefreeze-driedtoformulateLDPIs,andwerefoundtobesuitableforpulmonarydelivery.Astrongerinvivoanti-S.aureuspneumoniceffectoftheformulationwasfoundatatenfolddose,incomparisonwithunformulatedAGorpenicillin.TheLDPIssigni?cantlyreducedthetumornecrosisfactora(TNF-a)andinterleukin(IL)-1pro-in?ammatorycytokines.PhosphorylationofIjB-ainthenuclearfactor-jBpathwaywasalsoinhibitedtoanextraordinarydegree.Piazzinietal.[31]developedliposomesforthecentralnervoussystem(CNS)deliveryofAGbyaddingTween80aloneorincombinationwithdidecyldimethylammoniumbromideinordertomodifythesurfaceofthevesicles.TheabilityofliposomestoincreasethepermeabilityofAGwasevaluatedbyaparallelarti?cialmembranepermeabilityassay(PAMPA)andhCMEC/D3cells.Thesizeoftheliposomesrangedfrom(96.4±9.5)to(82.1±9.3)nm,andtheEErangedfrom44.7%±3.2%to47.5%±3.3%.Theliposomesshowedexcellentstabilityassuspen-sionsorfreeze-driedproducts.PAMPAandhCMEC/D3transportstudiesrevealedthatallthedevelopedliposomesincreasedthepermeabilityofAG,incomparisonwithfreeAG.Noalterationsincellviabilitywerefound.Themainuptakemechanismwascaveolae-mediatedendocytosis,andtheincreasedcellularinternalizationoftheformulationswasrelatedtothepositivecharge[31].2.2.4.SolidlipidnanoparticlesSLNs(Fig.3)areformulatedwithphysiologicallipids,whicharedispersedinwaterorinanaqueoussurfactantsolution.SLNsaretypicallyspherical,withanaveragediameterbetween10and1000nm.Thesolidlipidcoreisstabilizedbysurfactants(emulsi?ers)andformulatedusingfattyacids,monoglycerides,diglycerides,triglycerides,steroids,andwaxes.Allclassesofemulsi?erscanbeusedtostabilizethelipiddispersion,thuspreventingparticleagglomerationmoreef?ciently[15].AGencapsulatedintoSLNswasfoundtoenhanceantitumoractivityinBalb/cmicebecauseofanenhancementofbioavailabilityduetotheimprovementoftheAUC0–1andCmaxofAGincomparisonwithunformulatedAG.Asustained-releasepatternofAG-loadedSLNswasindicatedbyanincreasedvalueofTmax[32].AG-loadedSLNswithanaveragediameterof286.1nmandazetapotentialofà20.8mVweredevelopedtoimproveAGbioavail-ability.TheAGEEwas91.00%,whilethedrugloadingwas3.49%.Boththebioavailabilityandanti-hyperlipidemicef?cacyofAG-loadedSLNswereimprovedbyincreasingthestabilityandsol-ubilityofAGinthegastro-enterictract.ThebioavailabilityofAGloadedinSLNwasincreasedto241%incomparisonwithunformu-latedAG[33].AG-loadedSLNswereformulatedusingCompritol888ATOandBrij78.TheEEwas92%.TheSLNsshowedexceptionalphysicalandchemicalstabilityat4and25°Cafterstorageforonemonth.TheSLNswerealsostablewhendispersedinhumanserumalbuminandplasma.TheinvitroreleaseofAG-loadedSLNsatphysiologicalpHwasprolongedandsustained.TheSLNs’abilitytocrosstheBBBwasevaluatedinvitrobythePAMPAtestandhCMEC/D3cellsinordertopredictthepassivetranscellularpermeability.TheSLNsimprovedthepermeabilityofAGincomparisonwithfreeAG,andthedatafromthetwotestswerecomparable.Intravenouslyadmin-istered?uorescentSLNsweredetectedinbrainparenchymaoutsideofthevessel,thusestablishingtheirabilitytocrosstheBBB[34].M.Casamontietal./Engineering5(2019)69–75732.2.5.NanoemulsionsandmicroemulsionsBothNEsandMEs(Fig.3)arenanoscaleemulsionscharacterizedbyhighstabilityandtransparency.However,theterms‘‘ME”and‘‘NE”arenotinterchangeable;anMEisanisotro-picliquidmixturethatformsspontaneouslyandisthermodynam-icallystable,whereasanNEisananoscaledispersionthatisobtainedbymeansofmechanicalforceandisonlykineticallystable.ThetwophasesofNEsandMEsmaybewater-continuousoroil-continuous.Inaddition,MEscanpresentbicontinuoussys-tems(alsocalledsponge-likesystems)[15,35].AnAG-loadedNEwasformulatedinordertoimproveoralbioavailabilityandprotec-tiveactionagainstin?ammatoryboweldisease.TheAG-loadedNEwaspreparedwithwater,ethanol,a-tocopherol,andCremophorEL.TheoptimizedAG-loadedNEwasstableat4and25°Cforthreemonths,andhadadropletsizeof(122±11)nmandaviscosityof28centipoise.AnexvivotestusinganevertedratgutsacindicatedthatthejejunumwastheoptimalsiteforAGloadedintheNE.AGactivitywas8.21and1.40timeshigherthanthevaluesobtainedwithAGsuspensionandAGethanolsolution,respectively.Thepharmacokineticresultsshowedarelativebioavailabilityof594.3%whentheAG-loadedNEwascomparedwithanAGsuspen-sion.Inaddition,theulcerindexandhistologicaldamagescoreofindomethacin-inducedintestinallesionsinmiceweresigni?cantlyreducedbypretreatmentwithAG-loadedNE[36].Anoil-in-water(O/W)MEloadedwithAGwasdevelopedusingisopropylmyristate,Tween80,ethanol,andwater.Themeandro-pletsizewas15.9nm,andthesolubilityofAGwas8.02mgámLà1.Theformulationwasstable,withahigheranti-in?ammatoryeffectandbioavailabilitythanAGtablets;itdisplayednoacuteoraltoxicity[37].AspecialNEloadedwithAGwaspreparedusinglayer-by-layertechnologyviaelectrostaticdepositionofchitosanoveralginate-encrustedO/WNEbymeansofultra-sonication.Thebeststabilitywasobtainedafter20minofsonication.Theparticlesizeofthemulti-layeredNEwasmeasuredtobewithintherangeof90.8–167.8nm,withazetapotentialbetween22.90and31.01mV.TheNEshowedastrategicreleasepatternwhenassessedinvitroinvarioussimulatedbiological?uids.Itshowedsigni?cantmodulationinaliverfunctiontest(alanineaminotransferase(ALT),alkalinephosphatase(ALP),aspartatetransaminase(AST),totalbilirubin(TBIL),directbilirubin(DBIL),andliverglycogen)andserumcytokines(IL-b,TNF-a,IL-10,andIL-6)whenassessedinvivoingalactosamine-lipopolysaccharide-intoxicatedmice,thusexhibitingsigni?cantlyimprovedhepatoprotection[38].2.2.6.GoldnanoparticlesFunctionalizedgoldNPs(Fig.3)aresmartvectorsforbiomedi-calapplicationsthatcancontrolgeometricalandopticalproper-ties.Theirlargesurfacecanbecoatedwithdiversemolecules(i.e.,targetingagents,drugs,andanti-foulingpolymers),makingthemversatilecarriers.Inparticular,thetargeteddeliveryofdrugsisoneofthemostencouragingmedicinalusesofgoldNPs[39].EngineeredgoldNPsloadedwithAG,withasphericalgeometryof14nmandapolydispersionindex(PDI)valueof0.137,weredeveloped.Theyexhibitedresilientanti-leishmanialactivity,againstbothwild-type(IC50of(19±1.7)mmoláLà1)andsodiumstibogluconate(IC50of(55±7.3)mmoláLà1)/paromomycin(IC50of(41±6.0)mmoláLà1)-resistantstrains.CompletemacrophageuptakeofAGgoldNPsoccurredwithin2hofexposure,andthecyto-toxicitywassigni?cantlylowerthanthatofamphotericin-B[40].2.2.7.NanocrystalsandnanosuspensionsMaetal.[41]designedafast-dissolvingnanocrystal-basedsoliddispersiontoenhancethedissolutionofAG.Thenanodispersionwasformulatedusingvariousratiosofhydroxypropylmethylcellulose(HPMC)andthreesuperdisintegrantexcipients:sodiumcarboxymethylstarch,mannitol,andlactose.ThedispersionwiththehighestconcentrationofHPMCexhibitedthemostrapidAGdis-solutionproperties,probablyduetotheenhancedwettability.Theperformanceofthesuper-disintegrantsatalevelof20%,incombina-tionwith25%HPMC,wastested.Theformulationdevelopedwith15%sodiumcarboxymethylstarch,15%HPMC,and10%lactoseimprovedthedissolution(drugloadingwasupto67.83%±1.26%).Theinvivopharmacokineticsstudiesrevealedasigni?cantlyenhancedbioavailabilityofAGincomparisonwithunformulatedAG.Ananodispersionwaspreparedanditsperformancetested.Thedissolutionrate(85.87%),Cmax((299.32±78.54)ngámLà1),andAUC0–1((4440.55±764.13)mgá(háL)à1)oftheAGnanodispersionweresigni?cantlyhigher(P<0.05)incomparisonwithcrudeAG.TheAUC0–1wasthreetimeshigher[42].AGnanosuspensionscon-tainingglycyrrhizinhadameanparticlesizeof487nmandexhib-itedexcellentperformanceincomparisonwiththoseobtainedwithtrehalose.ThedissolutionpercentageoftheseAGnanosuspen-sions(99.87%)wassigni?cantlyincreasedincomparisonwiththatofunformulatedAG(42.35%)[43].Inanotherstudy,anAGnanosuspensionwaspreparedusingD-a-tocopherylpolyethyleneglycol1000succinate(TPGS,asur-factantthatinhibitsP-glycoproteinfunction)andsodiumlaurylsulfate.Themeanparticlesizewas(244.6±3.0)nmandthere-dispersibilityindexwas113%±1.14%(n=3).Increaseddissolutionbehaviorwasfound;inaddition,aCaco-2cellmonolayertestrevealedthatthemembranepermeability(Papp)ofAGinthenanosuspensionwassigni?cantlyhigherthanthoseofunformu-latedAGorAGnanosuspensionswithoutTPGS(P<0.01).Further-more,thenanosuspensioncontainingTPGSexhibitedsigni?cantlyhigherCmaxandAUC0–1(P<0.01).Astudyusingcarrageenan-inducedpawedemademonstratedthatanAGnanosuspensioncontainingTPGSwasmoreeffectiveandproducedagreaterincreaseintheserumlevelsofnitricoxide(NO),IL-1,andTNF-a(P<0.01)andinsuperoxidedismutase(SOD)activity(P<0.05)incomparisonwithunformulatedAG[44].Nanosuspensionswereformulatedwith3%AG,5%poloxamer188,0.1%sodiumtauroursodeoxycholate,or0.05%sodiumdeoxycholate,with0.4mmzirconiumoxidepearls.Thesenanosuspensionsshowedahexagonalmorphologyandaparticlesizeof300nm,butnochangeinthecrystallinehabitus.Asigni?cantincreaseinsaturationsolubilitywasfound,resultingincompletereleasewithin0.25h.Thelyophilizedformulationusingmannitol(5%)asacryoprotectanthadrespectablephysicalandchemicalstabilityduringthesix-monthstorageperiod.PharmacokineticandtissuedistributionstudiesrevealedthattheAGwasmainlydistributedintheliverandwasrapidlyeliminatedfromtheblood[45].3.ConclusionsMicrocarriersandnanocarriersareatremendouslyimportant?eldofresearchinbioactiveconstituents,whichincludenaturalproductsandherbalextracts.Theuseofthesetechnologieshasalreadyhadasigni?cantimpactonmanyareasofmedicinebyallowingappropriatetherapeutictreatmentsofcertainessentialdrugs(principallyantitumorandantiparasiticdrugs).Inadditiontoitsnumerouspotentialactivities,AG,whichisthecharacteristicconstituentofAndrographispaniculata(Burm.f.)Nees,hasalowbioavailability,limitedbiodistributionandlocalization,lackofsta-bilityingastrointestinalenvironments,andveryshortbiologicalhalf-life.RemarkableresultshavebeenobtainedinimprovingAGbioavailability,target-tissuedistribution,andef?cacyusingmicro-andnano-formulationsbasedonAG;theseachievements
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