Nanotechnology Formulations for Antibacterial Free Fatty Acids and Monoglycerides

19 Pages·2016·2.85 MB·English

molecules
Review
Nanotechnology Formulations for Antibacterial Free
Fatty Acids and Monoglycerides
JoshuaA.Jackman,BoKyeongYoon,DanlinLiandNam-JoonCho*
SchoolofMaterialsScienceandEngineeringandCentreforBiomimeticSensorScience,
NanyangTechnologicalUniversity,50NanyangDrive,Singapore637553,Singapore;
[email protected](J.A.J.);[email protected](B.K.Y.);[email protected](D.L.)
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:PeterJ.Rutledge
Received:1February2016;Accepted:23February2016;Published:3March2016
Abstract: Freefattyacidsandmonoglycerideshavelongbeenknowntopossessbroad-spectrum
antibacterialactivitythatisbasedonlyticbehavioragainstbacterialcellmembranes. Consideringthe
growingchallengesofdrug-resistantbacteriaandtheneedfornewclassesofantibiotics,thewide
prevalence, affordable cost, and broad spectrum of fatty acids and monoglycerides make them
attractiveagentstodevelopforhealthcareandbiotechnologyapplications. Theaimofthisreview
istoprovideabriefintroductiontothehistoryofantimicrobiallipidsandtheircurrentstatusand
challenges,andtopresentadetaileddiscussionofongoingresearcheffortstodevelopnanotechnology
formulationsoffattyacidsandmonoglyceridesthatenablesuperiorinvitroandinvivoperformance.
Examplesofnano-emulsions,liposomes,solidlipidnanoparticles,andcontrolledreleasehydrogels
arepresentedinordertohighlightthepotentialthatliesaheadforfattyacidsandmonoglyceridesas
next-generationantibacterialsolutions. Possibleapplicationroutesandfuturedirectionsinresearch
anddevelopmentarealsodiscussed.
Keywords: antimicrobial lipid; fatty acid; monoglyceride; nanotechnology; emulsion; liposome;
solidlipidnanoparticle;hydrogel
1. Introduction
Antibioticresistanceisoneofthemostseriouspublichealthissuesintheworld,sparkedinpart
bytheoveruseofantibioticsinmedicineandagriculture[1]. Inthefaceofmultidrug-resistantbacteria,
manyantibioticsarelosingeffectiveness,andthereisgrowingrecognitionthatapost-antibioticera
isapproaching[2]. Antibioticsareakeysectorofthepharmaceuticalindustry,asevidencedbyhigh
annual expenditures of up to US $10.7 billion in the United States alone [3,4]. The majority of the
expenditurecomesfromoutpatientdrugprescriptions[4]. Thewidespreadprescriptionofantibiotics,
especiallyintheoutpatientsetting,inevitablyresultsintheriseofmultidrug-resistantbacterialstrains.
Inrecentyears,therehasbeengrowingrecognitionoftheriseinmultidrug-resistanceamongvarious
bacterial strains as a major public health crisis [5]. Antibiotic-resistant bacterial strains have been
estimatedtoaffect2millionpatientsannuallyintheEuropeanUnionalone[6]. Meanwhile,inthe
UnitedStates,antibiotic-resistancecostsmorethanUS$20billionperyearaswellasanadditionalone
totwoweeksofinpatientcareperpatient,whichstrainstheexistingmedicalinfrastructure[6].
The antibiotic-resistance problem is further aggravated by the fact that new antibiotic drug
developmenthaslaggedbehindtheevolutionofantibiotic-resistantbacterialstrains. Fromthe1960s
upthrough2011,merelyfournewclassesofantibioticsweresuccessfullydevelopedandmarketed[6].
A2013studyrevealedthatonlyfourlargepharmaceuticalcompanieshaveactiveR&Dprograms
todevelopnewantibiotics,ascomparedto20companiesinthe1980s[3]. Asidefromthescientific
difficultyindevelopingnewantibiotics,thelackofnewantibioticshasbeenattributedtoaperceived
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lackofpotentialprofitduetocompetitionfromlowcost,off-patentgenericdrugsandtheshort-course
nature of antibiotic treatment. Historically unfavorable regulations and policies by government
agencies such as the United States Food and Drug Administration (FDA) have also been cited as
afactorwhichdiscouragesthedevelopmentofnewantibiotics[7]. Thereareincreasingdemands
fornarrow-spectrumantibioticswithfocusedactivityagainstspecificbacteriainordertomitigate
thechanceforantibiotic-resistantbacterialstrainstoemerge[8]. Broad-spectrumantibacterialswith
targetsthathaveveryhighbarrierstogeneratingresistancemutationsarealsoingreatdemand.
Recently, there has been serious attention directed to this issue because the number of
drug-resistant bacteria, or so-called super bacteria, continues to rise unabated [9,10]. There is
even growing discussion about the end of the antibiotic era altogether. In order to encourage the
developmentofnewantibiotics,theFDAhascreatedanewproductcategorycalledtheQualified
InfectiousDiseaseProduct(QIDP)[11]. Drugsinthiscategoryhaveaspecialdesignationfromthe
FDA,whichshortenstheapprovalreviewprocessandincreasesthetime(anadditional5years)for
exclusivemarketing. Thesebenefitsareimportantbecauseitmeansthatnewantibioticscanreach
patientsmorequickly,andthereismoreeconomicmotivationforpharmaceuticalcompaniestodevelop
newantibiotics. Inturn,therehasbeenapositiveriseinthenumberofnewantibioticsinthepasttwo
years[12]. However,nearlyallofthesenewlyapprovedantibioticsareinfactderivativesofexisting
antibioticsandshareoverlappingmechanismsofaction.Hence,theproblemofdrug-resistancequickly
emergingisnotavoidedbutratherprolonged,andthereisaneedforantibioticsagainstnovelbacterial
targets,especiallythosewithveryhighbarrierstomutation.
Inthisregard,membrane-activeantibacterialagentsholdsignificantpromise[13].Importantly,the
developmentofresistantbacterialstrainsagainstmembrane-activecompoundshaslowfrequency
becausethereisahighbarriertomutationofthebacterialcellenvelope[14].Membrane-activepeptides
emergedasanattractivecontenderandcanhavepotentantibacterialactivity[15]. Therearemany
academicstudiesonmembrane-activeantibacterialpeptidesandafewcandidateshaveevenreached
clinical trials (with at least one in Phase III trials) in the late 90s [16,17]. However, a particularly
stringentFDAapprovalprocessatthetimeledtothelackofapprovalforpeptidecandidatesofthat
era,andtherewasaconcomitantpushinthebiotechnologyindustryatlargetomovebeyondpeptide
therapeutics[18]. Inaddition,therearecommontechnicalissueswithmostmembrane-activepeptides,
includingweakperformanceinphysiologicalsaltconditions[19],cationiccharacteroftenrendersthem
toxictohumancells[20],andtypicaldependenceonaminoacidsecondarystructurewhichissensitive
toenvironmentalconditions. Moreover,membrane-activepeptidescanbecostlytoproduce[17].
At the same time, there is already a documented solution—naturally abundant and low cost
freefattyacidsandmonoglycerides(so-calledantimicrobiallipids)withbroad-spectrumantibacterial
activity—whichhaslongbeenknown,yetwascastasideinfavorofsmallmoleculeantibioticsforthe
pastfewdecades[21]. Withthecurrentchallengesinantibioticdrugdevelopment,antimicrobiallipids
deserverenewedattentionandrepresentpotentialsolutionstotheproblemofdrug-resistantbacteria.
Thepotentialoffreefattyacidsforbiotechnologyapplicationshasbeenhighlightedinatleasttwo
reviewsinthepastsixyears[22,23]. However,arguablythegreatestpotentialforantimicrobiallipids,
includingfreefattyacids,liesinnanotechnologyformulationswhichtakeadvantageofthepotent
antibacterialpropertiesofthesecompoundswhileimprovingtheirpharmacologicalpropertiesand
providingsuperiordeliveryvehicles. Indeed,theseeffortsfallundertheconceptofnanoarchitectonics,
anemergingsetofdesignguidelinestoincorporatefunctionalmoleculesintoapplication-oriented
nanostructures [24–27]. In recent years, there have been extensive efforts to achieve the goal of
establishingnanotechnologyformulationsforantimicrobiallipids,yetthecollectionofresearchefforts
towardsthisgoalhasnotbeensummarized.
The aim of this review is to introduce antimicrobial lipids as a class of potent antibacterial
agentsandtohighlightemergingnanotechnologyformulationsoffattyacidsandmonoglycerides.
Anoverviewofantimicrobiallipidsisfirstprovided,followedbyadetaileddescriptionofformulation
strategies based on nano-emulsions, liposomes, solid lipid nanoparticles, and controlled release
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hydrogels. Finally,theapplicationpotentialofthenanotechnologyformulationsisdiscussedinthe
contextofdevelopingeffectivesolutionstodrug-resistantbacteria.
2. OverviewofAntimicrobialLipids
Antimicrobiallipidswerefirstrecognizedasacomponentofthehumanbody’sinnateimmune
system. Modern interest in innate antibacterial compounds started with the discovery of the
antibacterialenzymelysozymeintheearly1900s[28].Sincethen,thetherapeuticpotentialofadditional
antibacterialcompoundsfoundwithintheinnateimmunesystem,includingantimicrobiallipids,has
been validated. On the human skin surface, there are two main types of innate host molecules
thatcontributetoantibacterialactivity,namely,antimicrobialpeptidesandantimicrobiallipids[29].
Recentresearchhasrevealedthatantimicrobialpeptidesareproducedbydifferentclassesofhuman
skincellsandhumanskinprokaryoticmicrobiotaastheimmunesystem’sfirstlineofdefenseagainst
bacterialinfection. Astherapeutics,however,antimicrobialpeptidesarecostlytoproduceinsufficient
quantities[28]andhaveothertechnicaldrawbacksasmentionedintheprevioussection. Ontheother
hand, antimicrobial lipids are abundant in prodigious quantities and exhibit broad-spectrum and
strongantimicrobialactivityontheskinsurface[30].
2.1. BriefHistory
Theearliestreportsofantimicrobiallipidsoriginatedinthelate19thcenturywhenKochobserved
thatfreefattyacidsinhibitthegrowthofBacillusanthracis[21]. Thisfindingrepresentedasignificant
advanceinscientificunderstandingbehindthehistoricalroleofsoaps—alkalisaltsoffattyacids—as
disinfectantsandcleaningagents. Intheensuingdecades,numerousstudiesdemonstratedthatfatty
acidsinhibitorkillawidespectrumofpathogens,leadingtospeculationaboutpossibletherapeutic
applications[31]. Inthe1930sand1940s,Burtenshawandcontemporariesidentifiedthatantimicrobial
fattyacidsandotherlipidsarefoundonhumanskinandactasnaturaldisinfectantstoregulatetheskin
microbiome[32]. However,theexplorationofantimicrobiallipidswastemperedbythewidespread
clinicaluseofpenicillinbeginninginthelate1940s,whichusheredinthemoderneraofantibiotics
andledtodramaticimprovementsinhumanhealthcare[33].
2.2. ClassesofAntimicrobialLipids
Free fatty acids are a widely studied type of antimicrobial lipid, and are composed of a
carboxylicacidgroupandasaturatedorunsaturatedcarbonchain. Theyfunctionasmildsurfactants
whichperturbbacterialcellmembranes[34],causingeitherbacteriostaticorbactericidaleffects[22].
Partialsolubilizationofthebacterialcellmembranecanimpairmetabolicregulationand/orcellular
energyproduction,whichleadstoinhibitionofbacterialgrowth. Moreextensivesolubilization,or
membranelysis,cantriggercelldeath,whichoftenoccursonthetimescaleofminutes. Assuch,there
are multiple ways by which fatty acids affect bacterial cell membranes; as a result, there is a high
barriertobacterialstrainsdevelopingresistancemutations[35]. Bycomparison,antibioticstypically
inhibitenzymesinvolvedinspecificpartsofthebacteriallifecycle,therebyincreasingthelikelihood
ofresistantstrainsemerging.
A wide range of antimicrobial lipids have been discovered or synthesized, and possess a
spectrum of antibacterial efficacies and targets. The main approach to classifying antimicrobial
lipidshasbeenstructure-activityrelationshipserieswhereininvitrostudiesfocusonmeasuringthe
inhibitionofbacterialgrowthinordertodeterminetheeffectofpermutations,suchashydrocarbon
chain length or the numbers of degrees of unsaturation, within a family of antimicrobial lipids.
Kabaraandcolleaguesconductedpioneeringstudiesontheantibacterialpropertiesofmedium-chain
saturatedfattyacids[36–38]. Basedondetailedstudiesofsaturatedfattyacidswithchainlengths
between6and18carbons, lauricacidwasidentifiedashavingthemostpotentinhibitoryactivity
againstGram-positivebacteria[39]. The1-monoglyceridederivativeoflauricacidiscalledglycerol
monolaurateandwasdiscoveredtohavealowerminimuminhibitoryconcentrationthanlauricacid,
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Molecules 2016, 21, 305 4 of 18
albeitagainstanarrowerrangeofbacteria[40]. Bothlauricacid(LA)andglycerolmonolaurate(GML)
RareecoGgenniezreadl lAysR Seacfoeg (nGizReAdSA) sbyS athfee (UGnRiAteSd) Sbtyattehse FUoondit eadndSt DatreusgF AooddmainndistDrartuiognA adsm foiondis tardadtiiotnivaess f[o41o]d.
Tahded icthiveemsi[c4a1l ]s.trTuhcetucrheesm oifc LaAls atrnudc tGuMreLs oafreL pAreasnedntGedM iLn aFrigeuprree s1e, nantedd hiinghFliigguhrte th1,ea tnwdo hmigahinli gclhatssthese
otwf aonmtimaiincrcolabsisael sliopfidasn ttihmati carroeb wiaildlieplyid sstuthdaitedar: efrweeid faeltytys atuciddise da:nfdr emeofantotyglaycciedrsidaensd. monoglycerides.

Figure 1. Chemical structures of a representative free fatty acid and monoglyercide.
Figure1.Chemicalstructuresofarepresentativefreefattyacidandmonoglyercide.
2.3. Activity Spectrum
2.3. ActivitySpectrum
Antimicrobial lipids have demonstrated effectiveness against various drug-resistant bacteria,
Antimicrobial lipids have demonstrated effectiveness against various drug-resistant bacteria,
including methicillin-resistant Staphylococcus aureus (MRSA) [29]. Considering the challenges faced
includingmethicillin-resistantStaphylococcusaureus(MRSA)[29]. Consideringthechallengesfacedby
by traditional antibiotics, developing therapeutic strategies based on natural compounds which are
traditionalantibiotics,developingtherapeuticstrategiesbasedonnaturalcompoundswhicharepartof
part of the human innate immune system arsenal is highly advantageous. Antimicrobial lipids are
thehumaninnateimmunesystemarsenalishighlyadvantageous.Antimicrobiallipidsareparticularly
particularly attractive because of their wide abundance in nature, perceived acceptance (several
attractive because of their wide abundance in nature, perceived acceptance (several antimicrobial
antimicrobial lipids are considered as GRAS by the US FDA), and bactericidal properties against
lipidsareconsideredasGRASbytheUSFDA),andbactericidalpropertiesagainstdifferenttypesof
different types of microorganisms including algae, bacteria, fungi, protozoa, and virus [22,36,39].
microorganismsincludingalgae,bacteria,fungi,protozoa,andvirus[22,36,39]. Owingtoaunique
Owing to a unique mechanism of action, antimicrobial lipids are less prone to the evolution of
mechanismofaction,antimicrobiallipidsarelesspronetotheevolutionofdrug-resistantbacterial
drug-resistant bacterial strains, as compared to traditional antibiotics. Indeed, antimicrobial lipids
strains, ascomparedtotraditionalantibiotics. Indeed, antimicrobiallipidsinterferewithbacterial
interfere with bacterial cell membranes, and can cause cell lysis or a range of indirect effects hindering
cellmembranes,andcancausecelllysisorarangeofindirecteffectshinderingcellmetabolism[22].
cell metabolism [22]. As mentioned above, structure-function relationships have shown that optimal
Asmentionedabove,structure-functionrelationshipshaveshownthatoptimalbactericidalactivity
bactericidal activity is obtained with saturated antimicrobial lipids with 10- or 12-carbon long fatty
isobtainedwithsaturatedantimicrobiallipidswith10-or12-carbonlongfattyacidssuchaslauric
acids such as lauric acid, which has successfully exhibited antibacterial activity against a wide range
acid,whichhassuccessfullyexhibitedantibacterialactivityagainstawiderangeofclinicallyrelevant
of clinically relevant pathogens, including S. aureus and P. acnes [22,42]. Antimicrobial lipids with
pathogens,includingS.aureusandP.acnes[22,42]. Antimicrobiallipidswithsimilarchainlengths
similar chain lengths (10 to 14 carbons) also show potent antimicrobial activity [37]. Other unsaturated
(10to14carbons) also show potent antimicrobial activity [37]. Other unsaturated fatty acids such
fatty acids such as linoleic acid and oleic acid have shown strong antibacterial activity too [43].
as linoleic acid and oleic acid have shown strong antibacterial activity too [43]. Interestingly, in
Interestingly, in many cases, the activity of a particular compound will overlap but not be identical
manycases,theactivityofaparticularcompoundwilloverlapbutnotbeidenticaltothatofother
to that of other antimicrobial lipids [22]. These findings motivate a more detailed understanding of
antimicrobiallipids[22]. Thesefindingsmotivateamoredetailedunderstandingofthecorresponding
the corresponding mechanisms, yet such information has proven largely elusive.
mechanisms,yetsuchinformationhasprovenlargelyelusive.
The main focus of structure-activity relationships of fatty acids and monogylcerides has been
Themainfocusofstructure-activityrelationshipsoffattyacidsandmonogylcerideshasbeen
concentration-dependent measurement of bacterial growth inhibition. The specific interaction between
concentration-dependentmeasurementofbacterialgrowthinhibition. Thespecificinteractionbetween
antimicrobial lipids and bacterial cell membranes remains to be understood. Indeed, the lipids
antimicrobial lipids and bacterial cell membranes remains to be understood. Indeed, the lipids
present a spectrum of mechanistic behaviors (e.g., permeabilization, lysis, etc.), with the specific details
present a spectrum of mechanistic behaviors (e.g., permeabilization, lysis, etc.), with the specific
depending on the chemical structure and concentration of the lipid [44]. In a few cases, transmission
details depending on the chemical structure and concentration of the lipid [44]. In a few cases,
electron microscopy has been utilized in order to analyze fixed specimens of bacteria after treatment
transmissionelectronmicroscopyhasbeenutilizedinordertoanalyzefixedspecimensofbacteria
with high (5–10 mM) concentrations of certain fatty acids and monoglycerides [45]. However, there
aftertreatmentwithhigh(5–10mM)concentrationsofcertainfattyacidsandmonoglycerides[45].
have been very few direct investigations of the membrane destabilization process caused by
However, there have been very few direct investigations of the membrane destabilization process
antimicrobial lipids. As presented in Figure 2, recent studies employing model membrane systems
causedbyantimicrobiallipids. AspresentedinFigure2,recentstudiesemployingmodelmembrane
called supported lipid bilayers have revealed that free fatty acids and monoglycerides can induce
systems called supported lipid bilayers have revealed that free fatty acids and monoglycerides
different kinds of morphological changes in the membrane [46,47]. A physicochemical explanation
caninducedifferentkindsofmorphologicalchangesinthemembrane[46,47]. Aphysicochemical
based on the lipid charge and critical micelle concentration was invoked in order to explain the
explanation based on the lipid charge and critical micelle concentration was invoked in order to
relationship between measured antibacterial activity and fundamental interactions with lipid
explaintherelationshipbetweenmeasuredantibacterialactivityandfundamentalinteractionswith
bilayers [47].
lipidbilayers[47].
Molecules2016,21,305 5of19
Molecules 2016, 21, 305 5 of 18

Figure 2. (a) Treatment of supported lipid bilayers with docosahexaenoic acid (DHA) induces three-
Figure 2. (a) Treatment of supported lipid bilayers with docosahexaenoic acid (DHA) induces
dimensional membrane morphological changes; (b) Schematic of DHA-induced, elongated lipid
three-dimensional membrane morphological changes; (b) Schematic of DHA-induced, elongated
structures on top of supported lipid bilayer. Top-down view of changes in the morphology of supported
lipid stlirpuidct buirlaeyserosn, intoclpudoinfgs u(cp) pmoermtebdranliep siodlubbiillaizyaetiro.nT (SoDpS-d troewatnmevnite)w; (do) fmcehmabnrganees biundtdhinegm (GoMrpLh ology
ofsupptroearttmedenlti)p; aidndb (iel)a myeerms,brinancelu fidbrinillgat(ioc)n m(LAem trbeartamneenst)o. lTuhbei sliczaalet iboanrs (iSnD alSl mtriecraotgmraepnhts) ;ar(ed 2)0m μemm. brane
buddinRgep(rGodMucLedt rweiathtm peernmt)i;ssaionnd fro(em) Rmefesm. [4b6r,a47n]e. fibrillation (LA treatment). The scale bars in all
micrographsare20µm.ReproducedwithpermissionfromRefs.[46,47].
2.4. Formulation Challenges
2.4. FormulDateisopnitCe hthaell penrogmesising antibacterial features of fatty acids and monoglycerides, there are technical
challenges which hinder the in vivo performance of compounds in the free form. One drawback that
Despitethepromisingantibacterialfeaturesoffattyacidsandmonoglycerides,therearetechnical
needs to be overcome is the poor solubility of antimicrobial lipids in aqueous buffer solutions [48].
challenWgehsilew ahnitcimhihcrinobdiealr ltihpeidisn cvainv obpe edrifsosormlveadn cine osoflcuotimonps ocuonndtasiniinngt hdeimfreetehyfol rsmulf.oOxindee d(DraMwSOba),c kthat
needsDtoMbSeOo ivs ea rsckoinm ireriitsantht aenpd otooxrics o[4l9u].b Oiltihtyero imfapnorttiamnitc fraoctboirasl toli poviderscoinmea qaruee tohue ssebnusiftfiveirtys otol ulitpiiodn s[48].
Whilecaonntciemntircartoiobni a(el.gli.p, cirditsiccaal nmibceelled icsosnoclevnetrdatiionn)s oanludt eionnvisrocnomnetnatianli nfagctodrism (ee.gth., ydlivsaulelnfot xcaidtieon(sD). MSO),
For these reasons, there has been strong motivation to develop improved formulations that overcome
DMSO is a skin irritant and toxic [49]. Other important factors to overcome are the sensitivity to
these challenges. In the following section, we describe nanotechnology solutions such as nano-emulsions,
lipid concentration (e.g., critical micelle concentration) and environmental factors (e.g., divalent
liposomes, solid lipid nanoparticles, and controlled release hydrogels which offer significant potential
cations). Forthesereasons,therehasbeenstrongmotivationtodevelopimprovedformulationsthat
to serve as carriers that not only deliver high concentrations of antimicrobial lipids, but also improve
overcotmhee ththereaspeecuhtiac lpleronpgeersti.esIn oft hanetifmolilcorowbiianlg lipseidcst.i on,wedescribenanotechnologysolutionssuchas
nano-emulsions,liposomes,solidlipidnanoparticles,andcontrolledreleasehydrogelswhichoffer
signific3a. Nntanpootteecnhtnioallotgoy sSetrravteegaiessc arriers that not only deliver high concentrations of antimicrobial
lipids,butalsoimprovethetherapeuticpropertiesofantimicrobiallipids.
3.1. Emulsions
3. NanotecAhqnuoeolougs yemSutrlsaitoengs iceosmposed of immiscible oil droplets in water are commonly prepared with
surfactants acting as emulsifiers to reduce tension between the oil and water phases and increase
3.1. Emeumlsuilosniosn stability. They can vary in size from the nanoscale to microscale. Owing to attractive
properties such as high stability and low viscosity, emulsions are regarded as potential drug delivery
Aqueousemulsionscomposedofimmiscibleoildropletsinwaterarecommonlypreparedwith
systems and are also widely studied for cosmetic, dermatology, food, and paint coating applications.
surfactNanonts-ioancitci snugrfaacstaenmtsu alrsei cfioemrsmtoonlrye cdhuosceen tbeencasuiosen thbeeyt waree elensst sheensoitiilvea ntod enwviartoenrmpenhtaasl ecosnadnitdionins,c rease
emulsisounchs atsa bthieli stoyl.utTiohne pyHc aannd vioanricy sitrnensgizthe, afnrod menhthanecen tahne opsecrmaleeabtoilitmy oicf rcoelslc maleem.brOanwesin. Sghotort aantdtr active
propermtieesdisuumc hchaasinh ailgcohhsotlas banildit ryelaantedd ldoewrivvatiisvceoss airtey ,aelsmo uuslesfiuoln ssurafraectraengtsa frodr epdreapsarpinogt eenmtuialsliodnrsu, gandde livery
systemcsana nddisaprlaeya alsnotibwaicdteerliyal satcutdiviietyd ifno rthceo semmeutliscio,nd esrtamtea. tToalobgley ,1 fosuomdm,aanridzeps atihnet ecxoiasttiinngg satpudpileisc ations.
reporting antimicrobial effects of emulsions. Al-Adham et al. first demonstrated that emulsions show
Non-ionicsurfactantsarecommonlychosenbecausetheyarelesssensitivetoenvironmentalconditions,
promise as antibacterial agents, with highly effective killing of several different types of bacteria,
suchasthesolutionpHandionicstrength,andenhancethepermeabilityofcellmembranes. Shortand
which was observed with rapid losses of bacterial cell viability upon treatment, supposed to have
mediumchainalcoholsandrelatedderivativesarealsousefulsurfactantsforpreparingemulsions,
arisen from membrane-active damage [50]. An example of bacterial cell membrane damage caused
andcabnyd eimspullasiyonasn itsi bpraecsteenrtieadl ianc Ftiivgiutrye i3n. theemulsionstate. Table1summarizestheexistingstudies
reportingantimicrobialeffectsofemulsions. Al-Adhametal. firstdemonstratedthatemulsionsshow
promiseasantibacterialagents,withhighlyeffectivekillingofseveraldifferenttypesofbacteria,which
wasobservedwithrapidlossesofbacterialcellviabilityupontreatment,supposedtohavearisenfrom
membrane-activedamage[50]. Anexampleofbacterialcellmembranedamagecausedbyemulsions
ispresentedinFigure3.
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Table1.Paststudiesinvolvingantibacterialpropertiesofmonoglycerideemulsions.
AntimicrobialLipid Organism Effects Reference
E.coli
Ethyloleate Ps.aeruginosa ‚BactericidalactivityagainstPs.aeruginosaandS.aureus,andinducedmembranealterationsagainstPs.Aeruginosa. [50]
S.aureus
C.jejuni
C.coli ‚~1mMmonocaprinemulsionscausedagreaterthan6-to7-log10reductioninviablebacterialcountofC.jejuni
within1min.
Monocaprin C.lari [51]
‚AntimicrobialactivityofmonocaprinemulsionsagainstC.coli,C.lari,Salmonellaspp.andE.coli.
Salmonellaspp.
wasdemonstrated.
E.coli
B.subtilis
Glycerolmonolaurate ‚IncreasedantibacterialactivityofGMLemulsionsversusfreeGMLagainstB.subtilisandE.coli. [52]
E.coli
‚DemonstratedantibacterialeffectofemulsionsagainstB.subtilis,withimprovedactivityinthepresenceof
Glycerolmonolaurate B.subtilis [53]
sodiumlactatesalt.
‚CompletelossofviabilityofE.coliorS.aureuscellswithin1mincausedbyhighlyconcentratedemulsions,and
E.coli
Glycerolmonolaurate slowkineticsobservedwith10-timesdilutedemulsions. [54]
S.aureus
‚Inducedreleaseofnucleicacidsduetobacterialmembranedamage.
B.subtilis
Glycerolmonolaurate ‚GMLemulsionsaremorepotentagainstB.subtiliswhereasfreeGMLisstrongeragainstE.coli. [55]
E.coli
S.maltophilia ‚GreaterantibacterialactivityofGMLemulsionsagainstS.maltophiliaversustheceftazidimeantibiotic,and
Glycerolmonolaurate [56]
E.coli enhancedactivitywithsodiumbenzoateasahydrotrope.
B.subtilis
‚CompletelossofviabilityofE.coli,S.aureusandB.subtiliscellswithin1mincausedbyGMLemulsions.
Glycerolmonolaurate E.coli [57]
‚Emulsionsdamagedbacterialcellwalls.
S.aureus
B.cereus
B.subtilis
E.faecalis
S.aureus ‚EnhancedantimicrobialactivitiesofemulsionsagainstGram-negativestrainsversus1-MAGsandoppositetrend
1-monoacylglycerol(1-MAG)ofcapric
M.luteus wasobservedwithGram-positivestains.
(C10:0),undecanoic(C11:0),lauric [58]
C.freundii ‚Bestantibacterialactivityagainstbothbacterialtypeswasobservedwith1-MAGC12:0emulsions.
(C12:0)„myristic(C14:0)acids
E.coli ‚10mg/Lconcentrationwasdeterminedtobethelimitformoderatetoxicity(40%–60%cellsurvival).
P.aeruginosa
S.entérica
S.marcescens
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Molecules 2016, 21, 305 7 of 18

Figure 3. Transmission electron microscopy micrographs of untreated (a) E. coli; (b) S. aureus; and (c)
Figure3. Transmissionelectronmicroscopymicrographsofuntreated(a)E.coli;(b)S.aureus;and
B. subtilis cells. Corresponding TEM mircrographs of microemulsion-treated (d) E. coli; (e) S. aureus;
(c)B.subtiliscells.CorrespondingTEMmircrographsofmicroemulsion-treated(d)E.coli;(e)S.aureus;
and (f) B. subtilis cells. Reproduced with permission from Ref. [57].
and(f)B.subtiliscells.ReproducedwithpermissionfromReference[57].
Expanding this research, there has been extensive research focused on exploring monoglycerides
Easx psuarnfadcitnagnttsh fiosrr pesreeparacrhin,gt heemruelhsiaosnsb.e Cenonesxidteenrisnigv ethraets meaornchogfloyccuersieddeso anree xnponlo-iroinngic msuornfaocgtalnytcse, rides
this choice is not surprising, but it is nonetheless interesting that all research on antibacterial
assurfactantsforpreparingemulsions. Consideringthatmonoglyceridesarenon-ionicsurfactants,
emulsions has strictly focused on monoglycerides without any reported studies on free fatty acids.
thischoiceisnotsurprising,butitisnonethelessinterestingthatallresearchonantibacterialemulsions
Thormar et al. demonstrated that, among different tested monoglycerides, monocaprin was the most
hasstrictlyfocusedonmonoglycerideswithoutanyreportedstudiesonfreefattyacids. Thormaretal.
active in killing C. jejuni, which is the leading cause of food-borne bacterial infections, and highly
demonstratedthat,amongdifferenttestedmonoglycerides,monocaprinwasthemostactiveinkilling
diluted monocaprin emulsions retained the ability to cause a >6- to 7-log10 reduction in the viable
C.jejuni,whichistheleadingcauseoffood-bornebacterialinfections,andhighlydilutedmonocaprin
bacterial count within 1 min [51]. However, other research has indicated that the diluted monocaprin
emulesmiounlssiroentsa einxhedibitth seiganbifiilciatyntt cohcaanugsees ian >st6ru-ctotu7re-l aongd1 0evreendtuucatliloy nloisnt atlhl eanvtiiambilcerobbaiaclt earcitaivlictyo.u Anst awn ithin
1 minal[te5r1n]a.tiHveo wsoeluvteiro,no twhiethr rgerseeaaterrc hsthabaislitiyn,d giclyacteerdolt hmaotntohleaudrialtue te(GdMmLo) neomcuaplsrioinnse mhauvles ipornosveenx hibit
signifisucapnertiochr aanndg easrein thset rmuoctsut rweidaneldy estvuedniteuda. lGlyMlLo setmaulllsainotnism wicerroeb sihaolwacnt itvo ihtya.vAe shiagnhearl taenrtnimatiicvreobsioallu tion
withagcrteivaitteyr tshtaanb iflrietye, GgMlyLce argoalimnsot nBo. lsauubtrialitse a(nGdM EL. c)oelim [5u2l,s5i3o,n55s],h aanvde aplrsoo vaepnpesaurpeder tioo rhaanved satrroentgheer most
antibacterial effects than the approved antibiotic ceftazidime [56]. GML emulsions can cause complete
widelystudied. GMLemulsionswereshowntohavehigherantimicrobialactivitythanfreeGML
loss of viability of bacterial cells in 1 min, while 10-fold diluted GML emulsions cause complete loss of
againstB.subtilisandE.coli[52,53,55],andalsoappearedtohavestrongerantibacterialeffectsthan
viability of bacterial cells within 10 min [54,57]. Acting as solubility enhancers, some salts, e.g., sodium
the approved antibiotic ceftazidime [56]. GML emulsions can cause complete loss of viability of
lactate and sodium benzoate, were found to enhance the antimicrobial effects of emulsions in optimized
bacterial cells in 1 min, while 10-fold diluted GML emulsions cause complete loss of viability of
preparations [53,56,57]. Further research indicated that the antimicrobial effects are related to the
bacterialcellswithin10min[54,57]. Actingassolubilityenhancers,somesalts,e.g.,sodiumlactate
interaction between the emulsions and bacterial membranes [54,57], after which there is a release of
and snoudclieuimc acbide nmzaoteartiea,l fwroemre infsoiuden dthet obaecntehriaanl cceelltsh aes aa nretsiumlti ocrf ombeimalberaffneec dtsisroufpetimonu alsnido ndyssifnunocptiotinm. ized
preparatiMonosst[ t5e3st,e5d6 ,s5p7e]c.ieFs uofr tbhoethr Grerasmea-prcohsitiinved aicnadt eGdratmh-antegthateivaen btaicmteirciar owbeirael sehfofwecnt stoa bree suresclaeptetidbleto the
interatoc tGioMnLb eemtwuleseionntsh. eFue met uall.s fioounnsda tnhdat bthaec tgerroiwalthm oefm Gbrarman-peoss[i5ti4v,e5 7B]., saufbtteilrisw whaisc ihnhthibeirteedi sbya GreMleLa seof
nucleeimcauclisdiomnsa mteorriae lsftrroonmgliyn sthidane tfhreee bGaMctLer, iwalhcileel ltshea sgarorwesthu lotfo Gfrmamem-nbergaantieved Eis.r cuopli twioans ainnhdibdityesdf ubny ction.
GML emulsions and free GML to similar extents [55]. Petra et al. systematically evaluated the effects
Most tested species of both Gram-positive and Gram-negative bacteria were shown to be
of monoglyceride emulsions versus free monoglycerides on a panel of Gram- positive and Gram-
susceptibletoGMLemulsions.Fuetal.foundthatthegrowthofGram-positiveB.subtiliswasinhibited
negative bacteria and determined that Gram-negative strains were more strongly inhibited by
by GML emulsions more strongly than free GML, while the growth of Gram-negative E. coli was
monoglyceride emulsions over free monoglycerides, while the opposite trend was observed with
inhibitedbyGMLemulsionsandfreeGMLtosimilarextents[55]. Petraetal. systematicallyevaluated
Gram-positive bacteria [44]. They also tested different monoglyceride emulsions and found that,
theeffectsofmonoglycerideemulsionsversusfreemonoglyceridesonapanelofGram-positiveand

Gram-negativebacteriaanddeterminedthatGram-negativestrainsweremorestronglyinhibitedby
monoglyceride emulsions over free monoglycerides, while the opposite trend was observed with
Gram-positive bacteria [44]. They also tested different monoglyceride emulsions and found that,
Molecules2016,21,305 8of19
amongthedifferentmonoglycerides,GMLemulsionshadthestrongestantibacterialactivityagainst
bothbacterialtypes. Despitethepromisingantibacterialactivity,emulsionscanalsoexhibittoxicity
tohumancells, andithasbeenreportedthat10mg/LofGMLemulsionskill40%–60%oftreated
human cells [58]. For this reason, emulsions appear to be best suited to applications such as food
preservativesandhighlightthechallengesofdevelopingtherapeuticallyviableformulationsforfatty
acidsandmonoglycerides.
3.2. Liposomes
Theoriginalmotivationbehindliposomaldrugdelivery(e.g.,liposomaldoxorubicin[59])was
greatertargetingspecificityandreducedtoxicityoftherapeuticagents. Successfulliposomedelivery
systemshavebeenreportedfortopicalandsystemicadministrationroutes. Inmanycases,liposomes
havebeendevelopedasrigidobjectstotargetinfectionsofthemononuclearphagocytesystemand
requirephagocyticuptake. However,rigidliposomeswithzwitterioniccharacterhavepoorefficacy
againstextracellularbacteria. Toaddressthischallenge,fusogenicliposomepreparationswithfluid
lipidbilayersandnegativemembranesurfacechargehavebeendeveloped[60]. Fusogenicliposomes
thatencapsulatetraditionalantibioticsexhibitstrongantibacterialactivity,evenwithonlysub-MIC
(minimum inhibitory concentration) concentrations of encapsulated antibiotic [61]. The fusogenic
liposomeshavebeenshowntofusewithbacterialcellmembranes[62],offeringapathwaytorescue
theantibacterialpropertiesofantibioticsagainstbacterialstrainsthathavebecomeresistanttothefree
formofthesaidantibioticdueto,e.g.,decreasedmembranepermeabilityorhighlyfunctionalbacterial
cellmembranetransporters[63]. Historically,Gram-positivebacteriahavebeenmoresusceptibleto
antibiotics,ascomparedtoGram-negativebacteria,whichpossessanadditionaloutermembrane. Asa
deliveryvehicle,fusogenicliposomeshaveacompetitiveadvantagebecausetheynotonlysupport
targeteddelivery,butalsoenablehighuptakeofantibacterialagentbybacteria.
Importantly, liposomal delivery vehicles can significantly reduce host cell toxicity and other
deleterious side effects of free fatty acids. As presented in this section, there is strong evidence
supportingthedevelopmentofliposomalfreefattyacidformulationstoreducebacterialloads,which
hasbeenreportedininvitroandinvivostudies,includingbothtopicalandsystemicadministration
routes. Smallliposomesloadedwithfattyacidsencapsulatedintheliposomalbilayercanfusewith
bacterialcellmembranes,leadingtoreleaseofhighlocalconcentrationsoffattyacidsintothebacterial
cellmembrane,whichresultsingrossmorphologicalchangesandenhancedmembranepermeability
thatcausesbacterialcelldeath[64]. Table2presentsasummaryofexistingstudieswhichhaveutilized
liposomalfreefattyacidstotreatbacterialinfections.
Yangetal. firstdemonstratedthatlauricacid-loadedliposomes(LipoLA)cansuccessfullytarget
withafusionmechanisminvolvingthebacterialcellmembraneandcompletelykillpreviouslyresistant
strainsofP.acnesat51µg/mLloadedLAconcentration[49]. Bycontrast,theMICoffreeLAisaround
80µg/mL.Inaddition,theantibacterialactivityofLipoLAwasdependentontheloadedamountof
LAperliposomeintheformulationwithoptimalactivityachievedat51%molefraction[49].Tofurther
explorethetherapeuticefficacyofLipoLA,Pornpattananangkuletal. evaluatedboththeinvitroand
invivo antibacterial activities. Invitro, the morphological changes caused by the fusion of LipoLA
with bacterial cell membranes and, invivo, the complete killing against P. acnes, achieved through
intradermalinjectionandtopicaladministrationat8mg/mLand2mg/mLofLipoLA,respectively,
confirmed the potency of LipoLA. Additional toxicity tests of LipoLA on mouse skin showed no
irritation,whichsupportsitstherapeuticpotentialagainstP.acnesinfection[65]. Meanwhile,therehas
alsobeenincreasingattentiontoexploringtheantibacterialactivityoflinolenicacid-loadedliposomes
(LipoLLA)againstH.pyloriinfection,whichisrelatedtoarangeofgastrointestinaldiseasesincluding
gastriccancer. Obonyoetal. identifiedthatLipoLLAhaspotentantibacterialactivityagainstH.pylori
inbothspiralandcoccoidforms,includingadiverserangeofantibiotic-resistantstrains. Aspresented
inFigure4,distortedmorphologiesofH.pyloriareobservedwithTEMaftertreatmentwithLipoLLA,
andthereisalsoasharpdecreaseintheminimumbactericidalconcentration(MBC)valuesforLipoLLA
versusfreeLLA.
Molecules2016,21,305 9of19
Table2.Paststudiesinvolvingantibacterialpropertiesofliposomalfattyacids.
AntimicrobialLipid Organism StudyDesign Effects Reference
Linolenicacid,
‚DemonstratedefficacyofLipoLLAwithMBCvalueof200µg/mL.
Stearicacid, H.pylori InVitro [65]
‚SignificanteffectofLipoLLAonincreasingoutermembranepermeabilityofH.pylori.
Oleicacid
‚Effectiveinkillingbothspiralandcoccoidformsofthebacteriabasedonmembranedisruption.
Linolenicacid H.pylori InVitro [66]
‚LipoLLAhashigherbarriertodevelopmentofdrug-resistantstrainsthanfreeLLA.
‚MBCvalueofLipoLAat51µg/mLagainstP.acnes.
Lauricacid P.acnes InVitro [50]
‚EstablishedimportanceofcriticalmolarfractionoffreefattyacidsinLipoFAs.
‚MBCvaluesforLipoLLAandLLAof65µg/mLand80µg/mL,respectively.
Linolenicacid H.pylori InVivo [67]
‚SignificantefficacyofLipoLLAinvivowithexcellentbiocompatibility.
‚Effectivetherapeuticefficacyof2mg/mLLipoLAintopicalformulation.
Lauricacid P.acnes InVivo [68]
‚NoirritationofnormalmouseskinbyLipoLLA.
S.aureus ‚12-foldincreaseininvitroefficacyofOAinliposomalformulationversusfreeOA.
Oleicacid InVivo [69]
(MRSA) ‚HighinvivoefficacyofLipoOAintreatmentofMRSAskininfections.
Molecules2016,21,305 10of19
Molecules 2016, 21, 305 10 of 18

Figure 4. (a) Fusion of fatty acid-loaded liposomes with bacterial cell membranes; (b) liposomal
Figure4.(a)Fusionoffattyacid-loadedliposomeswithbacterialcellmembranes;(b)liposomaldelivery
delivery to H. pylori-infected stomach lining; TEM micrographs of (c) control and (d) LipoLLA-treated
toH.pylori-infectedstomachlining;TEMmicrographsof(c)controland(d)LipoLLA-treatedH.pylori
H. pylori bacteria; (e) bacterial load of H. pylori in mouse stomach after treatment with different
bacteria;(e)bacterialloadofH.pyloriinmousestomachaftertreatmentwithdifferenttherapeutic
therapeutic agents. * p < 0.05, ** p < 0.001. Reproduced with permission from Refs. [64,66].
agents.*p<0.05,**p<0.001.ReproducedwithpermissionfromRefs.[64,66].
Importantly, LipoLLA has a higher barrier for inducing the mutation of drug-resistant strains even
Iamftepro trrteaantmtlye,nLt iwpiothL LeAquhivaasleanht iLgLhAer cboanrcreinetrrafotironins dfourc i1n0g dtahyes,m wuhtialeti oHn. opyfldorriu egv-ernetsuisatlalyn tacsqtruaiirneds even
resistance mutations to free LLA after only 3 days of treatment [68]. Thamphiwatana et al. also
aftertreatmentwithequivalentLLAconcentrationsfor10days,whileH.pylorieventuallyacquired
explored the therapeutic efficacy of LipoLLA against H. pylori infections in an in vivo mouse model.
resistance mutations to free LLA after only 3 days of treatment [68]. Thamphiwatana et al. also
It was demonstrated that LipoLLA has superior antibacterial activity against H. pylori over free LLA
exploredthetherapeuticefficacyofLipoLLAagainstH.pyloriinfectionsinaninvivomousemodel.
and conventional triple-therapy antibiotics based on measurements of bacterial burden in the mouse
ItwasdemonstratedthatLipoLLAhassuperiorantibacterialactivityagainstH.pylorioverfreeLLA
stomach after treatment [66]. Further, histopathological analysis of LipoLLA distribution in the
andconventionaltriple-therapyantibioticsbasedonmeasurementsofbacterialburdeninthemouse
mouse stomach showed that LipoLLA was able to penetrate the mucus layer of the mouse stomach
stomaacnhd areftmeraitnreeda timn tehnet l[a6y6e]r. fForu artt hleears,th 2i4s tho. pLaipthooLlLoAg iaclasol arnedaulycseids loevfeLlisp oof LpLroAindfliasmtrmibauttoiroyn ciyntotkhienems ouse
stomaarcihsinsgh ofrwomed Ht.h paytloLriip ionfLeLctAionw ians thabe lsetotmoapcehn leatyreart ewtihtheoumt uircruitsatliaoyn eorr ocfauthsiengm toouxisceitys taogmaiancsht and
rematienseteddi nmothuesel asytoemrafcohr caetlllse.a Isnt o2r4dher. tLoi pacohLieLvAe aa lgsroearteedr umceecdhalenvisetilcs uonfdperrostiannfldaimngm oaf ttohrey fucsyitoonk ines
arisinpgrofrcoesms, HJu.npgy loetr iailn. fpeecrtfioornminedt hdeetsatiolemda cchhalraayceterrwizaitthioonu tstiurrdiiteast ipornoobirncga uthsein igntteorxaicctiitoyn abgeatiwnesetnte sted
LipoLLA and related analogues against H. pylori through in vitro experiments [64]. The antibacterial
mousestomachcells. Inordertoachieveagreatermechanisticunderstandingofthefusionprocess,
activity of liposomal formulations of three different fatty acids in the C18 series, liposomal stearic
Jung et al. performed detailed characterization studies probing the interaction between LipoLLA
acid (LSA), liposomal oleic acid (LOA) and LLA, were tested, and it was concluded that LipoLLA
andrelatedanaloguesagainstH.pylorithroughinvitroexperiments[64]. Theantibacterialactivityof
had the most potent anti-H. pylori activity, with complete killing within 5 min. Moreover, further
liposomalformulationsofthreedifferentfattyacidsintheC18series,liposomalstearicacid(LSA),
investigation showed that both LipoLLA and oleic acid-loaded liposome (LipoOA) increased the
liposopmermaleoableiliictya ocfi dth(eL HO. Apy)loarni douLteLr Am,ewmebrreantee.s Itnetder,easntidngiltyw, thaes pcoernmceluabdielidty tohfa tthLe ipplaosLmLaA mheamdbrtahnee most
potenotf acenltlis- Htre.apteydlo rwiiathc tLivipitoyL,LwAi tshhocwoemdp al emteuckhi lglirnegatewr iitnhcirnea5sem thiann. tMhoosree torevaetre,df uwritthh eLripinovOeAs,t iagsa tion
showveidsuathlizaetdb boyt htraLnispmoisLsLioAn ealencdtroonl emicicraocsicdop-lyo (aTdEeMd) lmipicorsoogmrapeh(sL oifp Ho.O pyAlo)ri ibnaccrteeraisae (dcf. tFhigeupree 4r)m [6e4a].b ility
of the H. pylori outer membrane. Interestingly, the permeability of the plasma membrane of cells

treatedwithLipoLLAshowedamuchgreaterincreasethanthosetreatedwithLipoOA,asvisualized
by transmission electron microscopy (TEM) micrographs of H. pylori bacteria (cf. Figure 4) [64].