用于mRNA传递的脂质纳米颗粒 Lipid nanoparticles for mRNA delivery

1078|December2021|volume6/natrevmats

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LipidnanoparticlesformRNAdelivery

XuchengHou1,TalZaks2✉,RobertLanger3,4✉andYizhouDong

1✉

Abstract|MessengerRNA(mRNA)hasemergedasanewcategoryoftherapeuticagentto

preventandtreatvariousdiseases.Tofunctioninvivo,mRNArequiressafe,effectiveandstabledeliverysystemsthatprotectthenucleicacidfromdegradationandthatallowcellularuptake

andmRNArelease.Lipidnanoparticleshavesuccessfullyenteredtheclinicforthedeliveryof

mRNA;inparticular,lipidnanoparticle–mRNAvaccinesarenowinclinicaluseagainstcoronavirusdisease2019(COVID-19),whichmarksamilestoneformRNAtherapeutics.InthisReview,we

discussthedesignoflipidnanoparticlesformRNAdeliveryandexaminephysiologicalbarriersandpossibleadministrationroutesforlipidnanoparticle–mRNAsystems.Wethenconsiderkeypointsfortheclinicaltranslationoflipidnanoparticle–mRNAformulations,includinggood

manufacturingpractice,stability,storageandsafety,andhighlightpreclinicalandclinicalstudiesoflipidnanoparticle–mRNAtherapeuticsforinfectiousdiseases,cancerandgeneticdisorders.Finally,wegiveanoutlooktofuturepossibilitiesandremainingchallengesforthispromising

technology.

underclinicalevaluationforthepreventionandtreat-mentofvirusinfections,cancerandgeneticdiseases

7

–17

(Tables

1

,2

).

InthisReview,webrieflyoverviewrepresentativelipidnanoparticlesusedformRNAdeliveryanddescribekeystepsinthepreclinicaldevelopmentoflipidnano-particle–mRNAformulations,includingtheovercomingofphysiologicalbarriers,differentadministrationroutes,manufacturingandsafetyprofiles.Finally,wehighlightimportantexamplesoflipidnanoparticle–mRNAformu-lationsinclinicalstudiesandprovidefutureperspectivesforlipidnanoparticlesandmRNAtherapeutics.

DevelopmentoflipidsformRNAdelivery

In1976,nucleicacidswereencapsulatedanddeliveredinpolymericparticles

5

.Later,exogenousmRNAdeliveryintohostcellswasdemonstratedwithliposomes

22

,

23

(fig.

1

).Lipidsareamphiphilicmoleculesthatcontainthreedomains:apolarheadgroup,ahydrophobictailregionandalinkerbetweenthetwodomains.Cationiclipids,ionizablelipidsandothertypesoflipidhavebeenexploredformRNAdelivery(fig.

2

).

Cationiclipids.Cationiclipidshaveaheadgroupwith

permanentpositivecharges

11

,

14

.Forexample,1,2-di-O-octadecenyl-3-trimethylammonium-propane(DOTMA),aquaternaryammoniumlipid,hasbeenappliedformRNAdeliveryinmultiplecelltypes

24

,andwascommercializedasLipofectinincombinationwith1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE)

24

.1,2-dioleoyl-3-trimethylammonium-propane(DOTAP),abiodegradableanalogueofDOTMA,was

MessengerRNA(mRNA),whichwasdiscoveredbypio-neeringstudiesin1947–1961(ref.

1

),isatransientinter-mediatorbetweengenesandproteins.Bythelate1980s,investigationsofmRNAstructureandfunctionresultedinthedevelopmentofinvitro-transcribed(IVT)mRNA

2

.Sincethefirstproof-of-conceptanimalstudyin1990(ref.

3

),numerousstrategieshavebeenexploredtoamelioratetheinstabilityandimmunogenicityofIVTmRNA

2

,

4

.Additionally,advancesindrugdeliverysystemshaveexpeditedthepreclinicaldevelopmentofmRNAtherapeutics

5

–

17

,providingthebasisformRNAasanewclassofdrug(fig.

1

).

1DivisionofPharmaceutics&Pharmacology,Collegeof

Pharmacy,TheOhioStateUniversity,Columbus,OH,USA.

2Moderna,Inc.,Cambridge,MA,USA.

3DavidH.KochInstituteforIntegrativeCancerResearch,MassachusettsInstituteofTechnology,Cambridge,MA,USA.

4DepartmentofChemical

Engineering,MassachusettsInstituteofTechnology,

Cambridge,MA,USA.

✉e-mail:

tal.zaks@

;

rlanger@

;

dong.525@

/10.1038/

s41578-021-00358-0

mRNAhasshowntherapeuticpotentialinarangeofapplications,includingviralvaccines,proteinreplace-menttherapies,cancerimmunotherapies,cellularreprogrammingandgenomeediting

2

,

4

,

7

–

17

.Toachievetherapeuticeffects,mRNAmoleculeshavetoreachspe-cifictargetcellsandproducesufficientproteinsofinter-est.However,targeteddeliveryandendosomalescaperemainchallengingformRNAdeliverysystems,high-lightingtheneedforsafeandeffectivemRNAdeliverymaterials.

AvarietyofmaterialshavebeendevelopedformRNAdelivery,includinglipids,lipid-likematerials,polymersandproteinderivatives

7

–17

.Inparticular,lipidnanopar-ticleshavebeenthoroughlyinvestigatedandsuccessfullyenteredtheclinicforthedeliveryofsmallmolecules

18

,siRNAdrugs

18

andmRNA

19

–21

.Notably,twoauthorizedcoronavirusdisease2019(COVID-19)vaccines,mRNA-1273(refs

19

,20

)andBNT162b

21

,uselipidnanoparticlestodeliverantigenmRNA.Manyotherlipidnanoparticle–mRNAformulationshavebeendevelopedandare

NATurerevIewS|MATERIALSvolume6|December2021|1079

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19611961

mRNALipidnanoparticles

DiscoveryofmRNAanditsfunction1

Developmentofliposomes251

19651965

19691969

InvitrotranslationofisolatedmRNAinacell-freesystem252

Developmentofliposome–

1989

1989

Developmentofcationic

LNP–mRNAformulations24

1990

1990

LNPsencapsulatingsmallmolecules

(doxorubicinor

amphotericinB)were

approvedbytheFDA18

Developmentofliposome–mRNAformulationsas

influenzavaccine184

19931993

LNPsencapsulatingdaunorubicinwere

approvedbytheFDAandtheEMA18

1995

1995

1996

1996

2000

2000

LNPsencapsulatingverteporfinwere

approvedbytheFDA18

2001

2001

2005

20092009

LNPsencapsulatingvincristinewere

approvedbytheFDA18

20122012

ClinicaltrialofmRNAtherapeuticsusing

protamine–mRNAformulations

(NCT00204607)

LNPsencapsulatingirinotecanwere

approvedbytheFDA18

20152015

Firstin-humantestofpersonalizedmRNAcancervaccines209

LNPsencapsulatingcytarabinewere

approvedbytheFDA18

20172017

Onpattro(LNPs

encapsulatingsiRNA),thefirstsiRNAdrug,wasapprovedbythe

FDAandtheEMA18

20182018

20202020

1978mRNAformulations22,231978

FreemRNAtranslation

postintramuscularinjectioninmice3

Injectionofvasopressin

19921992

mRNAintoratbrainas

proteinreplacement

therapyfordiabetesinsipidus253

Injectionof

carcinoembryonicantigenmRNAintomousemuscle

asacancervaccine199

FirstclinicaltrialofmRNA-engineereddendriticcells(NCT00004211)

2005

Nucleoside-modifiedmRNAshowsreducedimmunogenicity182

ClinicaltrialofLNP–mRNAformulationsforcancer

20142014

immunotherapies(NCT02316457)

•ClinicaltrialofLNP–mRNAformulationsasinfluenzavaccines(NCT03076385)

•ClinicaltrialofLNP–mRNAformulationsforproteinreplacementtherapies

(NCT03375047)

•mRNA-1273andBNT162b(LNP–mRNAformulations)COVID-19mRNA

vaccinesobtainedauthorizationfromregulatoryagenciesinmultiple

countries

•ClinicaltrialofLNPformulations

deliveringgene-editingcomponentsforgeneticdisorders(NCT04601051)

Fig.1|TimelineofsomekeymilestonesformRNAandlipidnanoparticledevelopment.COVID-19,coronavirus

disease2019;EMA,EuropeanMedicinesAgency;FDA,UnitedStatesFoodandDrugAdministration;LNP,lipidnanoparticle

251

–253

.

alsostudiedformRNAdelivery

25

,andispartofthecommercialagentMegaFectin,togetherwithDOPEorcholesterol.DOTMAandDOTAPhavebothbeenappliedeitheraloneorcombinedwithothermaterialsformRNAdelivery

7

–

17

;forexample,spleen-targetedDOTMA–mRNAlipoplexes(RNA-LPX)havebeendevelopedassystemiccancervaccine

26

.Thesamefor-mulationhasalsobeendesignedasmRNAvaccineforthetreatmentofautoimmuneencephalomyelitis

27

.Thisvaccineinducestheproliferationofantigen-specific

CD4+regulatoryTcells,leadingtoenhancedimmu-nosuppressionandareductionofclinicalsymptomsinmousemodels

27

.DOTAP-basedcationicnanoemulsionscandeliverantigenmRNAagainstviral,bacterialandparasiticinfections

28

–

31

.Moreover,DOTAP–polymerhybridnanoparticlescandelivermRNAmoleculesforthetreatmentofcancer

32

–

37

,infections

38

–

41

andgeneticdisorders

42

.IncorporatingcarbonateapatiteinDOTAP-basedlipidnanoparticlesincreasestheinter-actionbetweentheparticlesandcellularmembranes

43

.

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Thedeliveryefficiencycanfurtherbeimprovedbycon-jugatingfibronectintothelipidnanoparticles,whichisacellularadhesionproteinacceleratingtheendocyticrate

44

.

Dimethyldioctadecylammoniumbromide(DDAB),aquaternaryammoniumlipid,cannotonlyfromcom-plexeswithmRNAbutalsostimulateinnateimmune

responses

45

,therebyservingasimmuneadjuvantformRNAvaccines

46

,

47

.DDABandDOPEconstitutethecommercialproductTransfectAce.Thecommer-cializedagentLipofectamineiscomposedofDOPEand2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoro-acetate(DOSPA),acationiclipidcontainingquaternary

Table1|Representativeclinicaltrialsoflipidnanoparticle–mRNAvaccinesagainstinfectionsandcancer

Name

Disease

Encodedantigen

Administrationroute

ClinicalTidentifier

Phase

Infections

mRNA-1273

SARS-CoV-2

Spike

i.m.

NCT04470427

III(EUA

andCMA)

BNT162b2

SARS-CoV-2

Spike

i.m.

NCT04368728

III(EUA

andCMA)

CVnCoV

SARS-CoV-2

Spike

i.m.

NCT04652102

III

LNP-nCoVsaRNA

SARS-CoV-2

Spike

i.m.

ISRCTN17072692

I

ARCT-021

SARS-CoV-2

Spike

i.m.

NCT04728347

II

ARCoV

SARS-CoV-2

Receptor-bindingdomain

i.m.

ChiCTR2000034112

I

mRNA-1440

InfluenzaH10N8

Haemagglutinin

i.m.

NCT03076385

I

mRNA-1851

InfluenzaH7N9

Haemagglutinin

i.m.

NCT03345043

I

mRNA-1893

Zikavirus

Pre-membraneandenvelopeglycoproteins

i.m.

NCT04064905

I

mRNA-1345

Respiratorysyncytial

virus

Fglycoprotein

i.m.

NCT04528719

I

mRNA-1653

Metapneumovirus

andparainfluenzavirustype3(MPV/PIV3)

MPVandPIV3Fglycoproteins

i.m.

NCT03392389

I

mRNA-1647

Cytomegalovirus

PentamericcomplexandBglycoprotein

i.m.

NCT04232280

II

mRNA-1388

Chikungunyavirus

Chikungunyavirus

antigens

i.m.

NCT03325075

I

CV7202

Rabiesvirus

Gglycoprotein

i.m.

NCT03713086

I

Cancer

mRNA-5671/

V941

Non-small-celllungcancer,colorectalcancer,pancreatic

adenocarcinoma

KRASantigens

i.m.

NCT03948763

I

mRNA-4157

Melanoma

Personalized

neoantigens

i.m.

NCT03897881

II

mRNA-4650

Gastrointestinal

cancer

Personalized

neoantigens

i.m.

NCT03480152

I/II

FixVac

Melanoma

NY-ESO-1,tyrosinase,MAGE-A3,TPTE

i.v.

NCT02410733

I

TNBC-MERIT

Triple-negative

breastcancer

Personalized

neoantigens

i.v.

NCT02316457

I

HARE-40

HPV-positivecancers

HPVoncoproteinsE6

andE7

i.d.

NCT03418480

I/II

RO7198457

Melanoma

Personalized

neoantigens

i.v.

NCT03815058

II

W_ova1

Ovariancancer

Ovariancancerantigens

i.v.

NCT04163094

I

CMA,conditionalmarketingauthorization;EUA,EmergencyUseAuthorization;HPV,humanpapillomavirus;i.d.,intradermal;

i.m.,intramuscular;i.v.,intravenous;KRAS,Kirstenratsarcoma2viraloncogenehomologue;MAGE-A3,melanomaantigenfamilyA;NY-ESO-1,NewYorkesophagealsquamouscellcarcinoma1;SARS-CoV-2,severeacuterespiratorysyndromecoronavirus2;

TPTE,putativetyrosine-proteinphosphatase.

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Table2|Representativeclinicaltrialsoflipidnanoparticle–mRNAtherapeuticsagainstinfections,

cancerandgeneticdisorders

Name

Disease

Encodedprotein

Administrationroute

ClinicalTrials.govidentifier

Phase

Infections

mRNA-1944

Chikungunyavirus

Antibodyagainst

chikungunyavirus

i.v.

NCT03829384

I

Cancer

mRNA2416

Solidtumours

OX40L

Intratumour

NCT03323398

II

mRNA-2752

Solidtumours

OX40L,IL-23andIL-36γ

Intratumour

NCT03739931

I

MEDI1191

Solidtumours

IL-12

Intratumour

NCT03946800

I

SAR441000

Solidtumours

IL-12sc,IL-15sushi,IFNαandGM-CSF

Intratumour

NCT03871348

I

Geneticdisorders

mRNA-3704

Methylmalonic

acidaemia

Methylmalonyl-CoAmutase

i.v.

NCT03810690

I/II

mRNA-3927

Propionicacidaemia

Propionyl-CoAcarboxylase

i.v.

NCT04159103

I/II

MRT5201

Ornithine

transcarbamylase

deficiency

Ornithinetranscarbamylase

i.v.

NCT03767270

I/II

MRT5005

Cysticfibrosis

Cysticfibrosistransmembrane

conductanceregulator

Inhalation

NCT03375047

I/II

NTLA-2001

Transthyretinamyloidosiswithpolyneuropathy

CRISPR–Cas9geneediting

system

i.v.

NCT04601051

I

CoA,coenzymeA;CRISPR–Cas9,clusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)–CRISPR-associatedprotein9;GM-CSF,granulocyte–macrophagecolony-stimulatingfactor;IFN,interferon;IL,interleukin;i.v.,intravenous.

ammoniumandspermine.LipofectamineprotocolshavebeenoptimizedtodelivermRNAindiversecelltypes,includingalveolarcells,cardiacmusclecellsandpluripotentstemcells

48

–

50

.2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethan-1-aminiumbromide(BHEM-Cholesterol)wasdevelopedbymodifyingtheheadstructureof3β-[N-(N,,N,-dimethylaminoethane)-carbamoyl]cholesterol(DC-Cholesterol)withhydroxylgroupstoimprovefusionwithcellularmembranes

51

.LipidnanoparticlescontainingBHEM-CholesterolhavebeenappliedtodelivermRNAencodingclusteredregularlyinterspacedshortpalindromicrepeats(CRISPR)–CRISPR-associatedprotein9(CRISPR–Cas9)andtumourantigens

52

,

53

.Ethylphosphatidylcholine(ePC)wassynthesizedbyintroducingathirdalkyloxygroupintophosphatidyl-cholinestoeliminatetheirnegativecharge.ePC-basedlipidnanoparticleshavebeenappliedformRNA-basedcancerimmunotherapies

54

,

55

andproteinreplacementtherapies

56

.

Ionizablelipids.Ionizablelipidsareprotonatedat

lowpH,whichmakesthempositivelycharged,buttheyremainneutralatphysiologicalpH(refs

7

,

11

,

14

).ThepH-sensitivityofionizablelipidsisbeneficialformRNAdeliveryinvivo,becauseneutrallipidshavelessinteractionswiththeanionicmembranesofbloodcellsand,thus,improvethebiocompatibilityoflipidnanoparticles

7

,

11

,

14

.Trappedinendosomes,inwhich

thepHislowerthanintheextracellularenvironment,ionizablelipidsareprotonatedand,therefore,becomepositivelycharged,whichmaypromotemembranedestabilizationandfacilitateendosomalescapeofthenanoparticles

7

,

11

,

14

Ionizablelipidsoriginallydevel-opedforDNAtransfection,suchas(2S)-2,5-bis(3-aminopropylamino)-N-[2-(dioctadecylamino)acetyl]pentanamide(DOGS;Transfectam)

57

,N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide(MVL5)

58

,DC-Cholesterol

59

andN4-cholesteryl-spermine(GL67)

60

,havealsobeenexploredformRNAdelivery

25

,61

–63

.

Theionizablelipid1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane(DLin-DMA)wasini-tiallysynthesizedforsiRNAdelivery

64

,anddeliv-eryefficacywasimprovedbymodificationofthelinkerandhydrophobicregions,resultingin2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA)

65

.FurtheroptimizationoftheamineheadgroupofDLin-KC2-DMAledto(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate(DLin-MC3-DMA;MC3),whichisakeydeliverycomponentofOnpattro,thefirstUnitedStatesFoodandDrugAdministration(FDA)-approvedsiRNAdrug

18

,

66

.MC3-basedlipidnanoparticleshavealsobeentestedformRNAther-apeutics,suchasproteinreplacementtherapies

56

,

67

–

72

andantiviraltherapies

73

–

75

.Incorporationofbiode-gradablelipidsimprovesthetolerabilityoflipidnano-particles,byallowingfastmetabolismwhileretainingmRNAdeliveryefficacy.Thebiodegradabilityoflipids

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canbeincreasedbyintroducingestermotifs;forexam-ple,introducingesterbondsinthelinkerandlipidictailsofMC3resultsinthelipiddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedio-ate(L319)

76

,whichshowsbetterdeliveryefficacyandfastereliminationfromtheliverandplasmainvivoincomparisonwithMC3(ref.

76

).Similarly,thebiode-gradablelipidsheptadecan-9-yl8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate(Lipid5)

77

,heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(unde-cyloxy)hexyl)amino)octanoate(LipidH(SM-102))

78

and((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)(ALC-0315)

79

havebetterinvivodeliveryefficacyandpharmacokineticsthanMC3.Ofnote,SM-102andALC-0315aretheionizabledeliv-erycomponentsinthemRNA-1273andBNT162bCOVID-19vaccines,respectively

17

.Biodegradablelipidscanalsobemadeofbothesteranddisulfidemotifs

80

–85

.Cleavageofthedisulfidebondsthendrivesanintrapar-ticlenucleophilicattackontheesterlinker,acceleratingtheirdegradation

80

–85

.

Acombinatoriallibraryhasbeendesignedthatcontainslipid-likematerialswithdifferenthydrophilicgroupsandmultiplelipidictails,highlightingthechem-icaldiversityofionizablelipids

86

.Manylipid-likemate-rials,suchas1,1′-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)(C12-200)

87

,tetrakis(8-methylnonyl)3,3′,3″,3川-(((methylazanediyl)bis(propane-3,1diyl))bis(azanetriyl))tetrapropionate(306Oi10)

88

and3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione(cKK-E12)

89

,havebeendevelopedtodelivermRNAmoleculesinvivo

90

–

100

.Forexample,cKK-E12-basedlipidnanoparticlesareappliedincancerimmunotherapies

94

,

95

andgenomeediting

96

.ReplacingthelipidicchainsofcKK-E12withalkenylaminoalcoholsresultsin3,6-bis(4-(bis((9Z,12Z)-2-hydroxyoctadeca-9,12-dien-1-yl)amino)butyl)piperazine-2,5-dione(OF-02),whichimprovesmRNAdeliveryefficacyinvivo,comparedwithcKK-E12(ref.

101

).FurtheralteringthelinkageofOF-02leadsto(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl)(9Z,9′Z,9″Z,9川Z,12Z,12′Z,12″Z,12川Z)-tetrakis(octadeca-9,12-dienoate)(OF-Deg-Lin)and(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(butane-4,1-diyl)(9Z,9′Z,9″Z,9川Z,12Z,12′Z,12″Z,12川Z)-tetrakis(octadeca-9,12-dienoate)(OF-C4-Deg-Lin),whichallowselectivedeliveryofmRNAintothespleen

102

,

103

.Thelipid-likematerialN1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide(TT3)candelivermRNAmol-eculesencodinghumanfactorIX

104

,CRISPR–Cas9(ref.

105

),aninterleukin-12(IL-12)replicon

106

andsevereacuterespiratorysyndromecoronavirus2(SARS-CoV-2)antigens

107

.Hexa(octan-3-yl)9,9′,9″,9川,9″″,9川″-((((benzene-1,3,5-tricarbonyl)ris(azanediyl))tris(propane-3,1-diyl))tris(azanetriyl))hexanonanoate(FTT5),whichisabiodegradableanalogueofTT3,furtherimprovestheinvivodeliveryefficacyofmRNAencodinghumanfactorVIIIandbaseeditingcomponents

108

.Inaddition,aseriesofaminoglycoside-derived

lipidshavebeensynthesizedasmRNAdeliverymaterials

109

–112

.

ZwitterionicionizablelipidscanalsobeappliedformRNAdelivery

56

,

113

–

116

;forexample,lipidscomposedofapH-switchablezwitterionandthreehydropho-bictailsassembleintoaconeintheendosomalacidicenvironment,enablingmembranehexagonaltrans-formationandallowingthemtoleavetheendosome.Thus,lipidnanoparticle–mRNAformulationsbasedonzwitterionicionizablelipidscanescapetheendosome,leadingtoefficientproteinexpressionandgenomeedit-inginvivo

114

.Inadditiontofunctioningasadeliverycomponent,lipidscanhavetherapeuticeffectssyner-gisticwithmRNA-encodedproteins

117

–

119

.Forexam-ple,lipidswithaheterocyclicamineasheadgroupcanactivatethestimulatorofinterferongenes(STING)signallingpathwayindendriticcells

117

.Theselipids,aspartofanmRNAvaccine,inducepotentcytolyticTlymphocyteresponsesandinhibittumourgrowthinmousemodels

117

.Paclitaxel-conjugatedlipidsencapsu-latingtumoursuppressormRNAcanbeappliedtointe-gratechemotherapyandgenetherapyfortriple-negativebreastcancer

118

.

Othertypesoflipid.Inadditiontocationicorionizable

lipids,lipidnanoparticle–mRNAformulationstypicallycontainotherlipidcomponents,suchasphospholip-ids(forexample,phosphatidylcholineandphosphati-dylethanolamine),cholesterolorpolyethyleneglycol(PEG)-functionalizedlipids(PEG-lipids)

7

,

14

,

17

.Theselipidscanimprovenanoparticleproperties,suchasparticlestability,deliveryefficacy,tolerabilityandbiodistribution

7

,

14

,

17

.Forexample,1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC),aphosphatidylcho-linewithsaturatedtails,hasameltingtemperatureof~54°CandacylindricalgeometrythatallowsDSPCmoleculestoformalamellarphase,whichstabilizesthestructureoflipidnanoparticles

120

.DSPChasbeenusedinthemRNA-1273andBNT162b2COVID-19vaccines

17

.DOPEisaphosphoethanolaminewithtwounsaturatedtails,whichhasameltingtemperatureof~30°Candaconicalshape

120

.DOPEtendstoadoptaninvertedhexagonalH(II)phase,whichdestabilizesendosomalmembranesandfacilitatesendosomalescapeoflipidnanoparticles

90

,120

.UsingDNAbarcode-labelledoligonucleotides,thedistributionofdifferentlipidnanoparticleformulationscanbeanalysedinahigh-throughputmannerinvivo

121

,forexample,toquantifytargeteddeliveryofnucleicacidsinmultipletissues

121

.Basedonthismethod,aseriesofphosphatidylcholinescontainingconstrainedadamantylgroupshasbeenexploredformRNAdelivery,includinganalysisofdistributionindifferentcelltypes

122

.

Cholesterolcanenhanceparticlestabilitybymodu-latingmembraneintegrityandrigidity

7

,14

,17

.Themolecu-largeometryofcholesterolderivativescanfurtheraffectdeliveryefficacyandbiodistributionoflipidnanopar-ticles.Forexample,cholesterolanalogueswithC-24alkylphytosterolsincreasetheinvivodeliveryefficacyoflipidnanoparticle–mRNAformulations

123

.Here,thelengthofthehydrophobictailsofthecholesterolana-logues,theflexibilityofsterolringsandthepolarityof

NATurerevIewS|MATERIALSvolume6|December2021|1083

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Cationiclipids

ONO

DOTMA

O

O

cN

O

O

DOTAP

Ionizablelipids

O

N

O

DLin-MC3-DMA

N

HOO

O

OO

ALC-0315

N

HOO

O

O

O

LipidH(SM-102)

OO

N

N

N

A2-Iso5-2DC18

O

HNO·S

八人ccSC

O

cNNiiOCS·S

OO·S

S

BAME-O16B

NiP

O

HCCOO·O

9A1P9

NH2

O

NH

HN

H2N

N

HN

O

O

DOSPAO

OO

O-P·OO

N

O

O

ePC

HOOH

OH

N

OH

N

N

N

N

OH

OH

C12-200

O

NH

HN人

N

OHN

OHOOH

cKK-E12

O

O

O

N

O

O

NH

OO

O

N

HN人

O

O

OF-Deg-Lin

O

O

O

O

N

N

N

O

O

O

O

306Oi10

O

N

HN

O

O

HN

N

N

NH

TT3

O

O

O

O

N

HN

O

O

O

O

O

O

O

N

N

HN

NH

O

O

FTT5

O

O

Othertypesoflipids

O

O

O

O

+-c·P·

O

O

O

OO

H3N

O

N

O

OO

O-P-OO

DOPE

DSPC

O

O

N

OO

O

O

O

45

O45

ALC-0159

O

PEG2000-DMG

◆

◆

◆

◆

H

H

H

H

OH

H

H

H

H

Br

O

O

H

IH

lIH

H

H

N

IH

H

-N

HO

O

NH

lIH

HO

NOH

HO

Cholesterol

BHEM-Cholesterol

DC-Cholesterol

ß-sitosterol

1084|December2021|volume6/natrevmats

Reviews

◀Fig.2|ChemicalstructuresoflipidsandlipidderivativesusedformRNAdelivery.

306Oi10,tetrakis(8-methylnonyl)3,3′,3″,3川-(((methylazanediyl)bis(propane-3,1