article nanoscale V21 noyellow - Royal Society of · PDF file3 Table of content Materials, general methods and cell culture 21 Synthesis of the pH-sensitive cationic switchable lipids

SupportingInformationCationicSwitchableLipids:pH-TriggeredMolecularSwitchforsiRNADelivery

Warren Viricel1, Steve Poirier2, Amira Mbarek1, Rabeb Mouna Derbali1, Gaetan Mayer1,2,

JeanneLeblond1*

1FacultyofPharmacy,UniversityofMontreal,P.O.Box6128,DowntownStation,Montreal,

QC,Canada

2 Laboratory ofMolecular Cell Biology,Montreal Heart Institute,Montreal, H1T 1C8, QC,

Canada

*[email protected]

Electronic Supplementary Material (ESI) for Nanoscale.This journal is © The Royal Society of Chemistry 2016

2

Tableoffigures

SchemeS1.SynthesispathwayofthecationicswitchablelipidsCSL1andCSL3 6

SchemeS2.SynthesispathwayofthecationicswitchablelipidCSL2 7

SchemeS3.SynthesispathwayofthenegativecontrolcationiclipidCSL4 8

SchemeS4.Protonationinducedconformationalchangeofthecationicswitchablelipids

CSL3andCSL4(negativecontrollipid) 9

FigureS5.PhysicochemicalcharacterisationofLNP/siRNAcomplexesmadebymanualextrusion 10

FigureS6.ForwardtransfectiononHeLa/GFPcellswithCSL3andCSL4LNP 11

FigureS7.CSL3/siRNA-Alexa647andfreesiRNA-Alexa647deliveryonHeLacells 12

FigureS8.Flowcytometryprofilesofuptakestudieswithendocytosisinhibitors 13

FigureS9.StabilitystudyofCSL3LNPmadebymicrofluidicmixing 14

FigureS10.QuantificationofbiodistributionofsiRNA-Cy5encapsulatedintoCSL3LNP 15

TableS11.siRNAsequencesusedinthisstudy 16

FigureS12.1HNMRand13CNMRspectrumsofcompoundCSL1 17




3

Tableofcontent

Materials,generalmethodsandcellculture 21

SynthesisofthepH-sensitivecationicswitchablelipidsCSL1andCSL3 22

1.1Synthesisofcompounds1&2 22

1.2Synthesisofcompound3 22



1.5SynthesisofcompoundCSL1 24


SynthesisofthepH-sensitivecationicswitchablelipidCSL2 26







SynthesisofthenegativecontrolcationiclipidCSL4 29



Lipidnanoparticlepreparationbymanualextrusion 31

siRNAcomplexationandencapsulationefficiency 32

Physicochemicalcharacterizationoflipidnanoparticles 33

InvitrosiRNAtransfectionassays 34

Invitrocytotoxicityassay 35

IntracytoplasmicdeliveryofsiRNA 36

EndosomalentrapmentoftheCSL4-basedformulation 37

Lipid-mixingassay 38

4

InhibitionofGFPknockdownwithBafilomycinA1 39

Uptakeinhibitionbyflowcytometry 40

Lipidnanoparticlepreparationbymicrofluidicmixing 42

Biodistributionandex-vivoimaginginmice 43

Serumstabilitystudy 44

InvivoFactorVIIsilencinginmice 45

References 46

5

Abbreviations

AcOEt: ethyl acetate; C12E8: octaethylene glycol monododecyl ether; CSL: cationic switchable lipid; DCM:

dichloromethane; DIPA: diisopropylamine; DMEM: Dulbecco’s Modified Eagle’s Medium, DMF:

dimethylformamide; DMG-PEG2000: 1,2-dimyristoyl-sn-glycerol methoxypolyethyleneglycol 2000; DMSO:

dimethyl sulfoxide; DSPC: (1,2-distearoyl-sn-glycero-3-phosphocholine; DSPE-PEG2000: N-(carbonyl-

methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine; EDTA:

ethylenediaminetetraacetic acid; EIPA: 5-(N-Ethyl-N-isopropyl)amiloride; EMEM: Eagle’s Minimum Essential

Medium;EtOH:ethanol;FBS:fetalbovineserum;FVII:coagulationfactorVII;GFP:greenfluorescentprotein;

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LDLR: lowdensity lipoprotein receptor; LNP: lipid

nanoparticle;MeOH:methanol;PBS :phosphatebuffersaline;PCSK9:proproteinconvertasesubtilisin/kexin

type 9; PEG: poly(ethyleneglycol); POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; R18: octadecyl

rhodamine B; TBAF: tetra-n-butylammonium fluoride; TEAA: triethylammonium acetate; TFA: trifluoroacetic

acid;THF:tetrahydrofuran.

6

SchemeS1.SynthesispathwayofthecationicswitchablelipidsCSL1andCSL3

i) NaH, THF, 80°C, 0.5h ; ii) ICH3,NaH, THF, 40°C, 2h ; iii) 5-bromo-2-methoxyphenylboronic acid, Pd(PPh3)4,

Na2CO3, THF/H2O, 80°C, 48h ; iv) 1-dodecyne, PdCl2(PPh3)2, PPh3, TBAF, DMF/DIPA, 80°C, 16h ; v) H2, Pd/C,

EtOH,roomtemperature,4h.

Thesynthesisandcharacterizationofthecompoundsarefurtherdetailedbelow.

NBr Br

NH2

+

i)N

I

NBr Br

HNN

NBr Br

NNN

ii)

NBr Br

NN

and

1 (62%)

2 (15%)

34 : R = CH2CH2N(CH3)25 : R = CH3

iii)

N

NNR

OO

Br Br

N

NNR

OO

C12H25 C12H25

CSL1 : R = CH2CH2N(CH3)2CSL3 : R = CH3

iv) v)HI

7

SchemeS2.SynthesispathwayofthecationicswitchablelipidCSL2

i)NaH,DMF,roomtemperature,3h;ii)Boc2O,DCM,roomtemperature,2h;iii)ICH3,NaH,DMF,40°C,3h;iv)

5-bromo-2-methoxyphenylboronicacid,Pd(PPh3)4,Na2CO3,THF/H2O,80°C,48h ;v)1-dodecyne,PdCl2(PPh3)2,

PPh3, TBAF,DMF/DIPA, 80°C, 16h ; vi)H2, Pd/C, EtOH, room temperature, 16h ; vii) HCl 4M, dioxane, room

temperature,2h.


NBr Br

NO2

+

i)H2N

NH2

NBr Br

HNNH2

ii)

NBr Br

HNNHBoc

6 7

N

NNHBoc

OO

Br Br

iii)

NBr Br

NNHBoc

8

iv)

9

v)

N

NNHBoc

OO

C10H21C10H21

10

vi) vii)N

NNH2

OO

C12H25 C12H25

CSL2

8

SchemeS3.SynthesispathwayofthenegativecontrolcationiclipidCSL4

i) 3-bromophenylboronic acid, Pd(PPh3)4, Na2CO3, THF/H2O, 80°C, 24h ; ii) 1-dodecyne, PdCl2(PPh3)2, PPh3,

TBAF,DMF/DIPA,80°C,16h;iii)H2,Pd/C,EtOH,roomtemperature,4h.


NBr Br

NN

3

i)

N

NN

Br Br

N

NN

C12H25 C12H25

ii) iii)

11 CSL4

9

SchemeS4.Protonation inducedconformational changeof the cationic switchable lipids

CSL3andCSL4(negativecontrollipid)

(A) Upon protonation of the central pyridine ring in the cationic switchable lipid CSL3, the formation of

intramolecularhydrogenbondingbetweentheprotonatednitrogenandthetwomethoxymoieties leadsthe

conformation to freeze, destabilizing the lipid nanoparticle and provoking endosomal escape. This behavior

wasobservedinapreviouswork.1(B)ThenegativecontrolcationiclipidCSL4lacksthetwomethoxymoieties

requiredtoperformintramolecularhydrogenbondingwiththeprotonatedpyridine,andisthereforeunableto

freeze its conformation at endosomal pH values. Note that the pKapyr values of the central pyridine ring of

thesetwolipidsaresimilar(5.39and5.22),aspredictedinsilico(CSpKaTMsoftware,ChemSilicoLLC.).

N

N

OO

NH

NH+

OO

CSL3

N

NA

[CSL3 + H]+

N

N

H+

CSL4

NB

[CSL4 + H]+

pKapyr = 5.39

pKapyr = 5.22

NH

N

N

INTRAMOLECULAR HYDROGEN BONDING

CONFORMATIONAL CHANGE

NO INTRAMOLECULAR HYDROGENBONDING POSSIBILITIES

NO CONFORMATIONAL CHANGE

10

Figure S5. Physicochemical characterisation of LNP/siRNA complexes made by manual

extrusion

Physicochemical characterisation of the lipid nanoparticles encapsulating anti-GFP siRNA at different lipid

nitrogen/siRNA phosphate (N/P) ratios. Lipid nanoparticles were prepared using cationic switchable lipids,

DSPC,cholesterolandDSPE-PEG2000atamolarratioof50:10:37.5:2.5respectively.Size(A)andpolydispersity

index(B)ofthelipidnanoparticles/siRNAcomplexesweremeasuredbydynamiclightscatteringinOpti-MEM®.

Zetapotential(C)oflipidnanoparticles/siRNAcomplexesweremeasuredindextrose5%.siRNAencapsulation

efficiency(D)wasmeasuredusingaSYBR®Goldfluorescenceassay.

0

50

100

150

200

250

300

Ave

rage

Dia

met

er (n

m)

N/P 1 N/P 2 N/P 6 N/P 8N/P 4No siRNA

CSL1

CSL2

CSL3

CSL4

-50

-40

-30

-20

-10

0

10

20

30

40

50

60

Zeta

Pot

entia

l (m

V)

CSL1

CSL2

CSL3

CSL4


0.0

0.1

0.2

0.3

Pol

ydis

pers

ity In

dex


CSL1

CSL2

CSL3

CSL4

0

10

20

30

40

50

60

70

80

90

100

110

siR

NA

Ent

rapm

ent E

ffici

ency

(%)

N/P 1 N/P 2 N/P 6 N/P 8N/P 4

CSL1 CSL3 CSL4CSL2

A B

C D

11

FigureS6.ForwardtransfectiononHeLa/GFPcellswithCSL3andCSL4LNP

Forward transfection on HeLa/GFP cells with CSL3 and CSL4-based LNP (CSL, DSPC, cholesterol and DSPE-

PEG2000 at amolar ratio of 50:10:37.5:2.5 respectively –made bymanual extrusion). Gene knockdownwas

assayedbyflowcytometryaftera48hincubationperiod(n=3).Statisticalanalysisperformedwithtwo-tailed

Student’st-test:ns.p>0.05;***p<0.001.

Forward transfection was realized tomake sure that transfection efficiency of CSL3 and CSL4 LNP remains

similar,regardlessofthechosentransfectionmethod(reverseorforwardtransfection).

0%

25%

50%

75%

100%

125%R

elat

ive

GFP

exp

ress

ion

10 nM siRNA

***

25 nM siRNA

CSL3 CSL4Untransfectedcells

***

ns

RNAiMAX

12

FigureS7.CSL3/siRNA-Alexa647andfreesiRNA-Alexa647deliveryonHeLacells

2-hourincubationofHeLacellswithCSL3/siRNA-Alexa647LNP(CSL3,DSPC,cholesterolandDSPE-PEG2000ata

molar ratioof50:10:37.5:2.5 respectively–madebymanualextrusion)or free siRNA-Alexa647. Final siRNA-

Alexa647concentrationinthedishis50nM.Representativepicturesareshown.Scalebaris20µM.

13

FigureS8.Flowcytometryprofilesofuptakestudieswithendocytosisinhibitors

Representative flow cytometry profiles of cell uptake (1-hour incubation (green) or 6-hour incubation (blue)) of the CSL3-based lipid nanoparticles formulation

(CSL3/DSPC/cholesterol/DSPE-PEG2000 50:10:37.5:2.5 mol% - made by manual extrusion) encapsulating siRNA-Alexa488 (25 nM/well), in the presence or absence of

endocytosisinhibitors.HeLacellswereusedforthisstudy.Clathrin(Chlorpromazine–10µg/mL),caveolae(Genistein–200µM),clathrin/caveolae(Pitstop2–20µM)or

macropinocytosis(EIPA–50µM)endocyticpathwayswereinhibited.Experimentwasrealisedintriplicate.

14

FigureS9.StabilitystudyofCSL3LNPmadebymicrofluidicmixing

CSL3 LNP (CSL3/DSPC/cholesterol/DMG-PEG2000 50:10:37.5:2.5 mol% - made by microfluidic mixing) were

incubatedinPBSbuffercontaining0%(A),10%(B)or50%(C)FBS.

UponstorageinPBSat+4°C,CSL3-basedLNParestablefor>7days.Whenincubatedinbuffercontaining10%

FBS,LNPremainstablefor10hat37°C.Nevertheless,aggregationoccursatthe10-hourand1-hourtimepoint

in10%and50%FBSrespectively.ThispartialseruminstabilitycouldresultintheprematurereleaseofsiRNAin

thebloodstreamfollowingintravenousinjection,explainingtherelativelyhighfluorescentsignalobservedin

thekidneysinthebiodistributionstudy(seeFigureS10).

0.1 1 10 100 1000 100000

5

10

15

20

Size (nm)

Inte

nsity

(%)

Day 1Day 3Day 7

0.1 1 10 100 1000 100000

5

10

15

20

Size (nm)

Inte

nsity

(%)

0 h1 h

Aggregation

2 h3 h4 h6 h8 h

10 h

0.1 1 10 100 1000 100000

5

10

15

20

Size (nm)

Inte

nsity

(%)

0 h1 h

Aggregation

2 h

FBS

A

B

C

PBS

10% FBS

50% FBS

15

FigureS10.QuantificationofbiodistributionofsiRNA-Cy5encapsulatedintoCSL3LNP

Quantification (photon counts normalizedbyorganwetmass) of siRNA-Cy5biodistribution. siRNA-Cy5were

encapsulatedintoCSL3LNP(CSL3/DSPC/cholesterol/DMG-PEG200050:10:37.5:2.5mol%-madebymicrofluidic

mixing).Organswereharvestedandimaged4hoursaftertailveininjectionatasiRNA-Cy5doseof1.5mg/kg.

Experimentwasrealizedintriplicate. Statisticalanalysisperformedwithtwo-tailedStudent’st-test:ns.p>0.05;

*p<0.05;**p<0.01.

Brain Lungs Heart Spleen Liver Kidneys0

1000

2000

3000

4000

5000

10000

20000

Nor

mal

ized

phot

on c

ount

s / m

g of

tiss

ue

Free siRNACSL3/siRNA

ns

**

**

ns

ns

ns

16

TableS11.siRNAsequencesusedinthisstudy

siRNA Sensestrand Antisensestrand Modification OriginsiRNAGFP Notprovided Notprovided None Dharmacon(cat#P-

002048-01-20)

siRNA-Alexa647 Notprovided Notprovided 3’-AlexaFluor647onthe

sensestrand

QiagenAllStarsNegativeControlsiRNA(cat#

1027287)

siRNA-Alexa488 Notprovided Notprovided 3’-AlexaFluor488onthe

sensestrand

QiagenAllStarsNegativeControlsiRNA(cat#

1027284)

siRNA-Cy5 5’-Cy5-UAGCGACUAAACACAUCAAUU-3’ 5’-UUGAUGUGUUUAGUCGCUAUU-3’ 5’-Cy5onthesensestrand

Dharmacon(siGENOMENon-TargetingControl

siRNA)siRNAPCSK9 5’-GccuGGAGuuuAuucGGAAdT*dT-3’ 5’-UUCCGAAuAAACUCcAGGCdT*dT-3’ None Dharmacon

siRNAFVII 5’-GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCdT*dT-3’ 5’-GfUAAGAfCfUfUGAGAfUGAfUfCfCdT*dT-3’ None Dharmacon

2’-OMemodifiednucleotidesare in lowercase.2’-Fmodifiednucleotidesaredenotedby“f”.Phosphorothioate linkagesarerepresentedbyasterisks.siRNAPCSK9and

siRNAFVIIsequenceswereobtainedfromtheliterature.2,3

17

FigureS12.1HNMRand13CNMRspectrumsofcompoundCSL1

[ppm] 8 6 4 2

[rel

] 0

5

1

0 1

5

7.67

527.

6698

7.25

807.

1396

7.13

427.

1187

7.11

327.

0038

6.90

446.

8834

3.83

82

3.55

063.

5321

3.51

28

2.62

952.

6155

2.59

742.

5783

2.34

27

1.65

681.

6383

1.62

151.

6026

1.58

36M

1.3

776

1.24

80M

1.1

882

0.88

910.

8727

0.85

52

1.77

96

2.02

331.

9238

2.07

65

6.00

00

3.93

38

7.97

57

11.6

313

4.29

31

39.1

965

6.38

73

N

NN

OO

C12H25 C12H25

N

CSL1

[ppm] 160 140 120 100 80 60 40 20

[rel

] 0

2

4

6

8

1

0 1

2

155.

8853

154.

9556

152.

0981

135.

3025

131.

4294

130.

1919

128.

9072

111.

4169

106.

3863

77.3

295

77.0

116

76.6

936

56.0

625

55.9

139

48.6

109

45.5

745

35.1

183

31.9

115

31.6

118

29.6

958

29.6

837

29.6

402

29.6

272

29.5

883

29.4

499

29.3

485

22.6

761

14.1

048

N

NN

OO

C12H25 C12H25

N

CSL1

18


[ppm] 6 4 2

[rel

] 0

5

1

0 1

5

7.62

917.

6247

7.25

867.

1601

7.15

557.

1391

7.13

467.

0411

6.90

156.

8804

3.85

153.

8232

3.70

59

3.10

46

2.60

822.

5892

2.56

94

M 1

.640

91.

6205

1.60

541.

5871

1.56

83M

1.3

720

1.29

321.

2458

M 1

.183

10.

8883

0.87

210.

8545

1.72

99

1.78

641.

7807

2.10

84

6.02

651.

5946

3.00

19

4.06

14

4.05

11

36.6

474

6.00

00

N

NNH2

OO

C12H25 C12H25

CSL2

[ppm] 160 140 120 100 80 60 40 20

[rel

] 0

5

1

0 1

5 2

0

154.

9576

154.

3002

154.

2331

135.

6021

131.

1364

129.

8030

127.

8747

111.

6380

106.

3076

77.3

507

77.0

328

76.7

148

56.0

014

38.5

071

35.0

572

31.9

147

31.6

243

29.6

914

29.6

461

29.5

786

29.4

301

29.3

526

22.6

783

14.1

080

N

NNH2

OO

C12H25 C12H25

CSL2

19


[ppm] 8 6 4 2

[rel

] 0

5

1

0 1

5

7.57

377.

5697

7.25

807.

2184

7.21

437.

1975

7.19

346.

9925

6.94

306.

9219

3.85

793.

7868

3.76

853.

7498

3.16

262.

9022

2.88

342.

8652

2.62

312.

6040

2.58

452.

5185

M 1

.662

31.

6267

1.61

071.

5923

1.57

36M

1.5

554

M 1

.367

01.

2441

M 1

.198

20.

8843

0.86

830.

8509

1.97

65

2.01

21

2.06

821.

9945

6.00

002.

0826

3.04

86

2.05

81

4.15

295.

9790

4.22

82

38.3

534

6.05

81

N

NN

OO

C12H25 C12H25

CSL3

[ppm] 150 100 50

[rel

] 0

5

1

0 1

5

166.

7523

154.

9223

154.

5536

153.

4140

135.

7720

131.

0461

130.

5229

126.

1564

111.

7032

106.

1171

77.3

360

77.0

182

76.7

003

56.0

546

54.4

687

48.7

174

44.3

466

38.5

057

35.0

076

31.9

034

31.5

934

29.6

740

29.6

306

29.6

214

29.5

483

29.3

908

29.3

386

22.6

695

14.1

004

N

NN

OO

C12H25 C12H25

CSL3

20


[ppm] 8 6 4 2

[ *1e

9] 0

2

0 4

0 6

0

7.90

037.

8714

7.85

19

7.39

487.

3758

7.35

677.

2585

7.23

737.

2183

6.91

83

5.28

66

3.76

983.

7512

3.73

18

3.13

082.

8188

2.79

942.

7809

2.72

642.

7073

2.68

762.

5095

1.71

921.

7004

1.68

311.

6640

1.64

47M

1.4

206

1.26

10M

1.1

970

0.89

810.

8815

0.86

39

1.95

802.

0383

2.01

222.

0176

2.00

00

1.95

64

2.95

87

1.93

594.

0549

6.04

40

4.02

62

37.3

220

6.06

11

N

NN

C12H25 C12H25

CSL4

[ppm] 160 140 120 100 80 60 40 20

[ *1e

9] 0

1

00

200

3

00

167.

1477

158.

1596

154.

6213

143.

1806

140.

5238

128.

7649

128.

3602

127.

3531

124.

5693

102.

2703

77.3

543

77.0

364

76.7

185

54.6

234

48.5

619

44.4

699

38.1

473

36.1

278

31.9

219

31.6

029

29.6

945

29.6

464

29.5

864

29.4

373

29.3

578

22.6

870

14.1

201

N

NN

C12H25 C12H25

CSL4

21

Materials,generalmethodsandcellculture

AllsolventsandreagentswereobtainedfromSigma-Aldrich(Oakville,ON,Canada),FisherScientific(Ottawa,

ON,Canada),AlfaAesar (WardHill,MA,USA)andOakwoodChemical (WestColumbia, SC,USA).Anhydrous

tetrahydrofuran (THF) and dimethylformamide (DMF) were obtained from a Pure Solv PS-400-6 System

(InnovativeTechnology,Amesbury,MA,USA).Cholesterol,DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine)

and DSPE-PEG2000 (N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-

phosphoethanolamine, sodiumsalt)were suppliedbyAvanti®Polar Lipids (Alabaster,AL,USA).DMG-PEG2000

(1,2-dimyristoyl-sn-glycerol,methoxypolyethyleneglycol2000)waspurchased fromNOFAmericaCorporation

(WhitePlains,NY,USA).Chloroquine,genistein,chlorpromazineandresazurinsodiumsaltwereobtainedfrom

Sigma-Aldrich (Oakville,ON,Canada), 5-(N-Ethyl-N-isopropyl)-Amiloride (EIPA)wasobtained fromSantaCruz

Biotechnology(Dallas,TX,USA),Pitstop2TMwasobtainedfromAbcam®(Toronto,ON,Canada)andBafilomycin

A1wasobtainedfromAlfaAesar(WardHill,MA,USA).Allsolventsandreagentswereofanalyticalgradeand

were used as received. All liquid nuclear magnetic resonance spectra were recorded on a Varian 400 WB

spectrometer, using residual solvent peak for calibration. High-resolution mass spectroscopy analysis was

performedby theRegional Center forMass Spectrometry of theUniversity ofMontréal. Chemical reactions

weremonitoredbyLC-MS(Infinity1260+MS6120,AgilentTechnologies,Mississauga,ON,Canada).

HeLa/GFPcells (GFPReporterStableCell Line,CellBiolabs Inc.,SanDiego,CA,USA)andHeLacells (CCL-2TM,

ATCC®,Manassas,VA,USA)wereculturedinEagle’sMinimumEssentialMedium(EMEM,cat#30-2003,ATCC®)

supplemented with 10% Fetal Bovine Serum (Gibco, Burlington, ON, Canada). Huh-7 cells were cultured in

Dulbecco’sModifiedEagle’sMedium(DMEM,cat#319-005-CL,Wisent,Montreal,QC,Canada)supplemented

with 10% Fetal Bovine Serum (Gibco). Cells were incubated at 37°C under a water-saturated atmosphere

supplementedwith5%CO2.

StatisticalanalyseswereperformedusingPrism®6(GraphPadSoftware,LaJolla,CA,USA).

22

SynthesisofthepH-sensitivecationicswitchablelipidsCSL1andCSL3

1.1Synthesisofcompounds1&2

Ina250mLround-bottomflaskpreviouslypurgedwithargonwasaddedsodiumhydride (60%dispersion in

mineraloil,un-rinsed)(926mg/27.6mmol).AnhydrousTHF(130mL)wasadded,and2,6-dibromopyridin-4-

amine4(1736mg/6.89mmol)wasslowlydissolvedinthemixture.2-iodo-N,N-dimethylethan-1-amine(iodide

salt)5 (2253 mg / 6.89 mmol) was added and the mixture was stirred 0.5 hours at 80°C under argon. The

mixturewasthenslowlyquenchedwithMeOH,anddry loadedintocolumnchromatographyonsilicagel for

purification (hexane/AcOEt, fast gradient from 100:0 to 0:100 ; DCM/MeOH, gradient from 100:0 to 70:30)

usingaTeledyneIscoCombiFlash®purificationsystem.Thefinalproduct1wasobtainedasayellowsolid(1386

mg/62%)andthefinalproduct2wasobtainedasaslightlyorangesolid(395mg/15%).

1:1HNMR(400MHz,CDCl3)δ(ppm)2.22(s,6H),2.52(t,J=5.7Hz,2H),3.08(q,J1=6.2Hz,J2=5.0Hz,2H),

6.56 (s, 2H).13C NMR (100MHz, CDCl3) δ (ppm) 39.49, 44.86, 56.68, 109.92, 140.60, 156.02. Calc [M+H] =

321.95490;Exp[M+H]=321.95555;Error=-2.01ppm.

2:1HNMR(400MHz,CDCl3)δ(ppm)2.25(s,12H),2.42(t,J=7.2Hz,4H),3.37(t,J=7.2Hz,4H),6.58(s,2H).

13CNMR(100MHz,CDCl3)δ(ppm)45.80,49.22,55.91,108.80,141.06,155.22.Calc[M+H]=393.02840;Exp

[M+H]=393.02934;Error=-2.41ppm.

1.2Synthesisofcompound3

Ina250mLround-bottomflaskpreviouslypurgedwithargonwasaddedsodiumhydride (60%dispersion in

mineraloil,un-rinsed)(654mg/19.5mmol).AnhydrousTHF(50mL)wasadded,and1(786mg/2.43mmol)

wasaddedtothemixture.Methyl iodidewasadded(449mg/3.16mmol).Thereactionwasstirredat40°C

andwascarefullymonitoredbyLC-MSuntilentireconsumptionofthestartingmaterial(~2h).Themixturewas

thenslowlyquenchedwithMeOH,filtered(0.20µmPTFEfilter)anddryloadedintocolumnchromatography

on silica gel for purification (hexane/AcOEt, fast gradient from 100:0 to 0:100; DCM/MeOH, gradient from

100:0to80:20)usingaTeledyneIscoCombiFlash®purificationsystem.Thefinalproduct3wasobtainedasa

23

yellowoil(640mg/78%).1HNMR(400MHz,CD3OD)δ(ppm)2.30(s,6H),2.49(t,J=7.4Hz,2H),2.99(s,3H),

3.49 (t, J =7.3Hz,2H),6.77 (s,2H).13CNMR (100MHz,CD3OD)δ (ppm)37.19,44.41,49.10,54.80,108.68,

140.19,156.34.Calc[M+H]=335.97055;Exp[M+H]=335.97180;Error=-3.71ppm.


Ina50mLround-bottomflaskwasdissolved2 (127mg/0.32mmol)and5-bromo-2-methoxyphenylboronic

acid (170mg/ 0.74mmol) in 4 mL of THF. Na2CO3 (137mg/ 1.29mmol), previously dissolved in 4 mL of

distilledwater,wasaddedandtheflaskwaspurgedthreetimeswithargon.Pd(PPh3)4(3.7mg/0.003mmol)

wasaddedandtheflaskwaspurgedthreetimesagain.Themixturewasstirred48hoursat80°C.Thereaction

wasmonitoredbyLC-MSuntilentireconsumptionofthestartingmaterials.Themixturewasthendilutedwith

saturatedNa2CO3andextracted3timeswithdichloromethane(DCM).TheorganiclayerwasdriedoverMgSO4

and evaporated under vacuum. The crude product was purified by reverse phase preparative HPLC on an

AgilentZorbaxPrepHTEclipseXDB-C1821.2x150mm(5µm)column.MobilephaseAwascomposedofwater+

0.1%formicacidandmobilephaseBwascomposedofMeOH+0.1%formicacid.Themobilephasegradient

was:0min–15%B;6min–80%B;12min-80%Bfollowedbyacolumnre-equilibrationtimeof4min.

Compound4wasobtainedasawhitesolid(58mg/30%).1HNMR(400MHz,CD3OD)δ(ppm)2.48(s,12H),

2.79(t,J=6.5Hz,4H),3.67(t,J=6.5Hz,4H),3.86(s,6H),6.97(s,2H),7.05(d,J=8.9Hz,2H),7.50(dd,J1=8.9

Hz,J2=2.6Hz,2H),7.73(d,J=2.6Hz,2H).13CNMR(100MHz,CD3OD)δ(ppm)44.14,55.21,56.08,107.00,

112.41, 113.35, 130.81, 132.32, 133.06, 152.55, 154.18, 156.23. Calc [M+H] = 605.11213 ; Exp [M+H] =

605.11451;Error=-3.93ppm.


Ina100mLround-bottomflaskwasdissolved3(694mg/2.06mmol)and5-bromo-2-methoxyphenylboronic

acid (1189mg/ 5.15mmol) in 6mL of THF. Na2CO3 (873mg/ 8.24mmol), previously dissolved in 6mL of




24


andevaporatedundervacuum.ThefinalproductwaspurifiedusingaTeledyneIscoCombiFlash®purification

system equipped with a 30 grams reversed-phase C18 HP Gold column.Mobile phase A was composed of

water+0.1%formicacidandmobilephaseBwascomposedofMeOH+0.1%formicacid.Themobilephase

gradient was: 0 min – 20 % B ; 15 min – 90 % B followed by a column re-equilibration time of 4-5 CV.

Compound5wasobtainedasawhitesolid(492mg/47%).1HNMR(400MHz,CD3OD)δ(ppm)2.35(s,6H),

2.62(t,J=7.2Hz,2H),3.07(s,3H),3.59(t,J=7.3Hz,2H),3.84(s,6H),6.94(s,2H),7.02(d,J=8.8Hz,2H),7.48

(dd,J1=8.8Hz,J2=2.5Hz,2H),7.70(d,J=2.5Hz,2H).13CNMR(100MHz,CD3OD)δ(ppm)37.09,44.29,48.82,

54.84,55.17,106.68,112.36,113.35,130.40,132.39,133.07,153.30,154.21,156.25.Calc[M+H]=548.05428;

Exp[M+H]=548.05574;Error=-2.66ppm.

1.5SynthesisofcompoundCSL1

Compound4(80mg/0.132mmol)wasweightedina20mLmicrowavereactionvial.1-dodecyne(66mg/0.40

mmol)andPPh3(10.4mg/0.04mmol)wereadded.Driedtetra-n-butylammoniumfluoride(TBAF)(241mg/

0.92mmol)wasadded(previouslydriedunderhighvacuumfrom1.0MTBAFsolutioninTHF),followedby2

mLofanhydrousDMFand2mLofdiisopropylamine(DIPA).Thereactionvialwaspurged3timeswithargon.

PdCl2(PPh3)2wasadded(8.3mg/0.01mmol),thevialwaspurged3timeswithargonandsealed.Themixture

wasstirred16hoursat80°C.ThemixturewasthendilutedwithsaturatedNa2CO3andextracted3timeswith

DCM.TheorganiclayerwasdriedoverMgSO4andevaporatedundervacuum.Thecrudeproductwaspurified

by column chromatography on silica gel (DCM/MeOH, gradient from 100:0 to 70:30) using a Teledyne Isco

CombiFlash® purification system. The product was then dissolved in EtOH in a 100mL round-bottom flask.

Palladium(10wt%Pdbasis)onactivatedcharcoal(40mg)wasaddedandtheflaskwaspurged3timeswith

hydrogen.Themixturewasthenstirredfor4hoursatroomtemperatureunder1atmhydrogenpressure.The

solution was then filtered on a PTFE filter (0.20 µm) to remove the catalyst, and the organic phase was

evaporated under vacuum. The final productwas purified by reverse phase preparativeHPLC on anAgilent

Zorbax PrepHT Eclipse XDB-C8 21.2x100mm (5µm) column.Mobile phaseAwas composed ofwater + 0.1%

formicacidandmobilephaseBwascomposedofMeOH+0.1%formicacid.Themobilephasegradientwas:0

min – 30 % B ; 10 min – 95 % B ; 12 min - 95 % B followed by a column re-equilibration time of 4 min.

25

CompoundCSL1wasobtainedasawhitesolid(40mg/38%).1HNMR(400MHz,CDCl3)δ(ppm)0.87(t,J=6.6

Hz,6H),1.19-1.38(m,36H),1.62(quint,J=7.4Hz,4H),2.34(s,12H),2.58-2.63(m,8H),3.53(t,J=7.4Hz,4H),

3.84(s,6H),6.89(d,J=8.4Hz,2H),7.00(s,2H),7.12(dd,J1=8.4Hz,J2=2.3Hz,2H),7.67(d,J=2.3Hz,2H).13C

NMR (100MHz, CDCl3) δ (ppm) 14.10, 22.68, 29.35-29.68, 29.70, 31.62, 35.12, 45.57, 48.61, 55.91, 56.06,

106.39,111.42,128.91,130.19,131.43,135.30,152.10,154.96,155.89.Calc[M+H]=785.6667;Exp[M+H]=

785.66949;Error=-3.54ppm.


Compound5 (228mg/0.42mmol)wasweightedina20mLmicrowavereactionvial.1-dodecyne(207mg/

1.25mmol)andPPh3(33.7mg/0.125mmol)wereadded.Driedtetra-n-butylammoniumfluoride(TBAF)(760

mg/2.91mmol)wasadded(previouslydriedunderhighvacuumfrom1.0MTBAFsolutioninTHF),followed

by2mLofanhydrousDMFand2mLofdiisopropylamine (DIPA).The reactionvialwaspurged3 timeswith

argon.PdCl2(PPh3)2wasadded(26mg/0.037mmol),thevialwaspurged3timeswithargonandsealed.The

mixturewas stirred 16 hours at 80°C. Themixturewas thendilutedwith saturatedNa2CO3 and extracted 3

timeswithDCM.Theorganic layerwasdriedoverMgSO4andevaporatedundervacuum.Thecrudeproduct

was purified by column chromatography on silica gel (DCM/MeOH, gradient from 100:0 to 90:10) using a

Teledyne Isco CombiFlash® purification system. The productwas then dissolved in EtOH in a 100mL round-

bottomflask.Palladium(10wt%Pdbasis)onactivatedcharcoal(50mg)wasaddedandtheflaskwaspurged3

timeswith hydrogen. Themixturewas then stirred 4 hours at room temperature under a 1 atm hydrogen

pressure. The solutionwas then filtered on a PTFE filter (0.20 µm) to remove the catalyst, and the organic

phasewasevaporatedundervacuum.ThefinalproductwaspurifiedbyreversephasepreparativeHPLConan

AgilentZorbaxPrepHTEclipseXDB-C821.2x100mm(5µm)column.MobilephaseAwascomposedofwater+

0.1%formicacidandmobilephaseBwascomposedofMeOH+0.1%formicacid.Themobilephasegradient

was:0min–30%B;10min–95%B;12min-95%Bfollowedbyacolumnre-equilibrationtimeof4min.

CompoundCSL3wasobtainedasaslightlyyellowwax(134mg/44%).1HNMR(400MHz,CDCl3)δ(ppm)0.87

(t,J=6.4Hz,6H),1.20-1.37(m,36H),1.59(quint,J=7.4Hz,4H),2.52(s,6H),2.60(t,J=7.5Hz,4H),2.88(t,J=

7.5Hz,2H),3.16(s,3H),3.77(t,J=7.5Hz,2H),3.86(s,6H),6.93(d,J=8.4Hz,2H),6.99(s,2H),7.20(dd,J1=

8.4Hz,J2=2.2Hz,2H),7.57(d,J=2.2Hz,2H).13CNMR(100MHz,CDCl3)δ(ppm)14.10,22.67,29.34-29.63,

26

29.67,31.59,35.00,38.51,44.35,48.72,54.47,56.05,106.12,111.70,126.16,130.52,131.05,135.77,153.41,


SynthesisofthepH-sensitivecationicswitchablelipidCSL2


Ethylenediamine (640 mg / 10.64 mmol) was added dropwise to a suspension of sodium hydride (60%

dispersion inmineraloil,un-rinsed) (255mg/10.64mmol) inanhydrousDMF (20mL).2,6-dibromo-4-nitro-

pyridine6(2g/7.10mmol)wasintroducedbysmallamounts.Themixturewasstirredatroomtemperaturefor

3hours, thencarefullyquenchedwithMeOH,washedwithsaturatedNa2CO3andextractedwithAcOEt.The

organic layer was dried over MgSO4 and concentrated to afford a brown oil. The crude was purified by

chromatographyonsilicagel (DCM/MeOH,gradient from100:0 to80:20)usingaTeledyne IscoCombiFlash®

purificationsystemtoafford6(628mg/30%)asanorangeoil.1HNMR(400MHz,CD3OD)δ(ppm)2.80(t,J=

7.0Hz, 2H), 3.18 (t, J = 7.0Hz, 2H), 6.70 (s, 2H). 13CNMR (100MHz, CD3OD) δ (ppm) 40.81, 45.12, 110.61,



To a solution of6 (1.5 g / 5.08mmol) in DCM (25mL)was slowly added Boc2O (1.33 g / 6.10mmol). The

mediumwasstirredatroomtemperatureduring2handthenwashedwithsaturatedNa2CO3.Theorganiclayer

wasdecantedandtheaqueouslayerwasextractedtwicewithDCM.Thecombinedorganiclayersweredried

over MgSO4 and concentrated under reduced pressure to afford a brown oil. The crude was purified by

chromatographyonsilicagel(hexane/AcOEt,gradientfrom100:0to0:100)usingaTeledyneIscoCombiFlash®

purificationsystemtoafford7 (1.6g/79%)asanorangeoil.1HNMR(400MHz,CDCl3)δ(ppm)1.44(s,9H),

3.20(m,2H),3.38(m,2H),4.92(t,J=2.4Hz,1H),5.54(br,1H),6.54(s,2H).13CNMR(100MHz,CDCl3)δ(ppm)

28.26,39.58,44.65,80.43,109.69,140.66,156.24,157.40.Calc[M+H]=393.97603;Exp[M+H]=393.97785;

Error=4.6ppm.

27


Toasolutionof7(1.2g,3.04mmol)inanhydrousDMF(10mL)wasaddedsodiumhydride(60%dispersionin

mineraloil,un-rinsed)(153mg/4.56mmol)andmethyliodide(863mg,6.08mmol).Themixturewasstirred

at40°CandwasmonitoredbyLC-MSuntilentireconsumptionofthestartingmaterial(~3h).Themixturewas

thenslowlyquenchedwithMeOH,washedwithwaterandextractedwithDCM.Thecrudewasdryloadedinto

column chromatography on silica gel for purification (hexane/AcOEt, gradient from 100:0 to 0:100) using a

TeledyneIscoCombiFlash®purificationsystemtoafford8(907mg/73%)asanorangesolid.1HNMR(400MHz,

CDCl3)δ(ppm)1.42(s,9H),2.97(s,3H),3.29(m,2H),3.47(m,2H),4.70(br,1H),6.65(s,2H).13CNMR(100

MHz,CDCl3)δ(ppm)25.76,35.30,48.64,77.40,106.30,138.46,153.49,153.72.Calc[M+H]=407.99168;Exp

[M+H]=407.99221;Error=1.3ppm.


Ina50mLround-bottomflaskwasdissolved8 (240mg/0.59mmol)and5-bromo-2-methoxyphenylboronic

acid (340mg/ 1.47mmol) in 5 mL of THF. Na2CO3 (249mg/ 2.35mmol), previously dissolved in 5 mL of





and evaporated under vacuum. The crude was dry loaded into column chromatography on silica gel for

purification (hexane/AcOEt, gradient from 100:0 to 0:100) using a Teledyne Isco CombiFlash® purification

systemtoafford9(179mg/49%)asanorangesolid.1HNMR(400MHz,CDCl3)δ(ppm)1.37(s,9H),3.09(s,

3H),3.38(m,2H),3.60(m,2H),3.85(s,6H),4.79(br,1H),6.86(d,J=8.7Hz,2H),6.95(s,2H),7.44(dd,J1=8.7

Hz,J2=2.5Hz,2H),7.84(d,J=2.5Hz,2H).13CNMR(100MHz,CDCl3)δ(ppm)28.37,38.31,51.25,56.22,79.42,

106.69,113.22,113.29,131.01,132.44,133.75,153.62,156.11,164.74.Calc[M+H]=620.07541;Exp[M+H]=

620.07685;Error=2.3ppm.

28


Compound9 (270mg/0.34mmol)wasweightedina20mLmicrowavereactionvial.1-dodecyne(113mg/

1.30mmol)andPPh3(20mg/0.078mmol)wereadded.Driedtetra-n-butylammoniumfluoride(TBAF)(355mg

/2.60mmol)wasadded(previouslydriedunderhighvacuumfrom1.0MTBAFsolutioninTHF),followedby1


PdCl2(PPh3)2wasadded(21mg/0.030mmol),thevialwaspurged3timeswithargonandsealed.Themixture



bycolumnchromatographyonsilicagel (hexane/AcOEt,gradient from100:0 to0:100)usingaTeledyne Isco

CombiFlash®purificationsystemtoafford10(141mg/41%)asayellowoil.1HNMR(400MHz,CDCl3)δ(ppm)

0.87(t,J=6.8Hz,6H),1.16-1.34(m,28H),1.35-1.50(m,13H),1.58(quint,J=7.6Hz,4H),2.37(t,J=7.2Hz,

4H),3.05(s,3H),3.36(m,2H),3.54(m,2H),3.84(s,6H),4.69(br,1H),6.88(d,J=8.6Hz,2H),6.92(s,2H),7.35

(dd,J1=8.6Hz,J2=2.4Hz,2H),7.80(d,J=2.4Hz,2H).13CNMR(100MHz,CDCl3)δ(ppm)14.10,19.45,22.67,

28.27, 28.91-29.60, 31.89, 38.08, 38.21, 51.00, 55.80, 80.36, 88.83, 106.55, 111.27, 116.60, 130.66, 132.44,

134.66,155.42,156.32.Calc[M+H]=792.56738;Exp[M+H]=792.5681;Error=0.91ppm.


Compound10(141mg/0.179mmol)wasdissolvedinEtOHina100mLround-bottomflask.Palladium(10wt

%Pd basis) on activated charcoal (50mg)was added and the flaskwas purged 3 timeswith hydrogen. The

mixturewas then stirred 4 hours at room temperatureunder a 1 atmhydrogenpressure. The solutionwas

thenfilteredonaPTFEfilter (0.20µm)toremovethecatalyst,andtheorganicphasewasevaporatedunder

vacuum. The oily crude was dissolved in 4M HCl in dioxane (2 mL) and was stirred 2 hours at room

temperature.ThemixturewasthenslowlydilutedwithsaturatedNa2CO3andextracted3timeswithDCM.The

organiclayerwasdriedoverMgSO4andevaporatedundervacuum.Thefinalproductwaspurifiedbyreverse

phasepreparativeHPLConanAgilentZorbaxPrepHTEclipseXDB-C821.2x100mm(5µm)column.Mobilephase

Awascomposedofwater+0.1%formicacidandmobilephaseBwascomposedofMeOH+0.1%formicacid.

Themobilephasegradientwas:0min–30%B;10min–95%B;12min-95%Bfollowedbyacolumnre-

29

equilibrationtimeof4min.CompoundCSL2wasobtainedasawhitewaxysolid(80mg/64%).1HNMR(400

MHz,CDCl3)δ(ppm)0.87(t,J=7.0Hz,6H),1.18-1.37(m,36H),1.59(quint,J=7.5Hz,4H),2.59(t,J=7.8Hz,

4H),3.10(s,3H),3.71(m,2H),3.82(s,6H),6.89(d,J=8.4Hz,2H),7.04(s,2H),7.14(dd,J1=8.4Hz,J2=2.3Hz,

2H),7.63(d,J=2.3Hz,2H).13CNMR(100MHz,CDCl3)δ(ppm)14.11,22.68,29.35-29.69,31.62,31.91,35.06,

38.51,56.00,106.31,111.64,127.87,129.80,131.14,135.60,154.23,154.30,154.96.Calc[M+H]=700.57756;

Exp[M+H]=700.57702;Error=-0.8ppm.

SynthesisofthenegativecontrolcationiclipidCSL4


Ina50mLround-bottomflaskwasdissolved3(171mg/0.51mmol)and3-bromophenylboronicacid(234mg/

1.17mmol)in4mLofTHF.Na2CO3(215mg/2.03mmol),previouslydissolvedin4mLofdistilledwater,was

addedandtheflaskwaspurgedthreetimeswithargon.Pd(PPh3)4(6.0mg/0.005mmol)wasaddedandthe

flaskwaspurgedthreetimesagain.Themixturewasstirred24hoursat80°C.Thereactionwasmonitoredby

LC-MSuntilentireconsumptionofthestartingmaterials.ThemixturewasthendilutedwithsaturatedNa2CO3

andextracted3timeswithdichloromethane(DCM).TheorganiclayerwasdriedoverMgSO4andevaporated

undervacuum.ThefinalproductwaspurifiedbyreversephasepreparativeHPLConanAgilentZorbaxPrepHT

EclipseXDB-C1821.2x150mm(5µm)column.MobilephaseAwascomposedofwater+0.1%formicacidand

mobilephaseBwascomposedofMeOH+0.1%formicacid.Themobilephasegradientwas:0min–15%B;6

min–80%B;12min-80%Bfollowedbyacolumnre-equilibrationtimeof4min.Compound4wasobtained

asaslightlyyellowwaxysolid (139mg/56%). 1HNMR(400MHz,CD3OD)δ (ppm)2.80(s,6H),3.03(s,3H),

3.15(t,J=2.3Hz,2H),3.78(t,J=2.3Hz,2H),6.92(s,2H),7.33(t,J=7.7Hz,2H),7.52(d,J=7.6Hz,2H),7.95

(d,J=7.7Hz,2H),8.19(s,2H).13CNMR(100MHz,CD3OD)δ(ppm)38.13,43.68,47.46,54.19,104.01,123.58,

126.98, 131.04, 131.30, 132.77, 143.14, 156.30, 156.93. Calc [M+H] = 488.03315 ; Exp [M+H] = 488.03532 ;

Error=4.4ppm.

30


Compound11(139mg/0.28mmol)wasweightedina10mLmicrowavereactionvial.1-dodecyne(139mg/

0.83mmol)andPPh3(22mg/0.083mmol)wereadded.Driedtetra-n-butylammoniumfluoride(TBAF)(509mg

/1.95mmol)wasadded(previouslydriedunderhighvacuumfrom1.0MTBAFsolutioninTHF),followedby1


PdCl2(PPh3)2wasadded(17mg/0.025mmol),thevialwaspurged3timeswithargonandsealed.Themixture



by column chromatography on silica gel (DCM/MeOH, gradient from 100:0 to 90:10) using a Teledyne Isco

CombiFlash® purification system. The product was then dissolved in EtOH in a 100mL round-bottom flask.

Palladium(10wt%Pdbasis)onactivatedcharcoal(50mg)wasaddedandtheflaskwaspurged3timeswith

hydrogen.Themixturewasthenstirred4hoursatroomtemperatureundera1atmhydrogenpressure.The

solution was then filtered on a PTFE filter (0.20 µm) to remove the catalyst, and the organic phase was

evaporated under vacuum. The final productwas purified by reverse phase preparativeHPLC on anAgilent

Zorbax PrepHT Eclipse XDB-C8 21.2x100mm (5µm) column.Mobile phaseAwas composed ofwater + 0.1%

formicacidandmobilephaseBwascomposedofMeOH+0.1%formicacid.Themobilephasegradientwas:0

min – 30 % B ; 10 min – 95 % B ; 12 min - 95 % B followed by a column re-equilibration time of 4 min.

CompoundCSL4wasobtainedasacolorlessoil(45mg/24%).1HNMR(400MHz,CDCl3)δ(ppm)0.88(t,J=6.6

Hz,6H),1.20-1.42(m,36H),1.68(quint,J=7.5Hz,4H),2.51(s,6H),2.71(t,J=7.5Hz,4H),2.80(t,J=7.5Hz,

2H),3.13(s,3H),3.75(t,J=7.6Hz,2H),6.92(s,2H),7.22(d,J=7.6Hz,2H),7.38(t,J=7.6Hz,2H),7.86(d,J=

7.6Hz, 2H), 7.90 (s, 2H).13CNMR (100MHz, CDCl3) δ (ppm) 14.12, 22.69, 29.36-29.69, 31.60, 31.92, 36.13,

38.15,44.47,48.56,54.62,102.27,124.57,127.35,128.36,128.76,140.52,143.18,154.62,158.16,167.15.Calc

[M+H]=668.58773;Exp[M+H]=668.58994;Error=3.31ppm.

31

Lipidnanoparticlepreparationbymanualextrusion

Lipid nanoparticles for in vitro studies were prepared by manual extrusion, using cationic switchable lipids

(CSL), DSPC, cholesterol and DSPE-PEG2000 at a molar ratio of 50:10:37.5:2.5 respectively. For in vivo

experiments, lipid nanoparticles were prepared using microfluidic mixing (further detailed below). Stock

solutions (20-40mg/mL)ofcationicswitchable lipidsandcommercialco-lipidswereprepared inethanoland

storedat-80°Cbeforeuse.Thelipidstocksolutionswerecombinedina5mLround-bottomflaskatthedesired

molar ratio, and theethanolwas removedunder reducedpressure. The lipidic film thusobtainedwasdried

overnightunderhighvacuumtoremoveanyresidualsolvent.Thelipidicfilmwashydratedonavortexmixer

with 1mL of sterile 5% dextrose in water. The lipid suspensionwas then subjected to stepwise extrusions

throughpolycarbonatemembranes(200and100nm–9passagespermembrane)usingaLiposoFastmanual

extruder (Avestin Inc.,Ottawa,ON,Canada)heatedat60°C.Thecationic switchable lipidamountpresent in

each preparation was quantified via HPLC-UV/MS (1260 Infinity, Agilent Technologies) against a calibration

curveofcationicswitchablelipid(25-250µg/mL;fromethanolstocksolution).

32

siRNAcomplexationandencapsulationefficiency

Stocksolutionsoflipidnanoparticle(madebymanualextrusion)andsiRNAweredilutedinsterile5%dextrose

atappropriateconcentrations,dependinguponthelipidnitrogen/siRNAphosphate(N/P)ratiodesired(unless

indicatedotherwise,aN/Pratiovalueof4wasusedforalltheexperiments).siRNAsolutionwasaddedtothe

lipidnanoparticlesolution,followedbybriefvortexing.Complexationwasthenrealizedduring15minutesat

50°Cundervigorousvortexing(1200rpm)inaLabnetVortempTM56.

Forquantificationoftheencapsulationefficiency,lipidnanoparticlesandsiRNAwerepreparedforafinalsiRNA

concentrationof100nM.ASYBR®Gold(ThermoScientific)assaywasusedtoquantifythesiRNAencapsulation

efficiencyof the formulations.Aftercomplexation (15minutes,50°C,vigorousvortexing), thesolutionswere

centrifugedat20000gfor30minutes.UnencapsulatedfreesiRNAinthesupernatantwasquantifiedagainsta

calibration curve of siRNA (2-100 nM), using the SYBR®Gold fluorescent dye (λex/em = 495/537) and a Safire

microplate reader (Tecan, Seestrasse, Switzerland). siRNA encapsulation efficiency (%) was calculated as

follows:

!! % = 100(()) − ,-./012(13(4564-2(-(4ℎ3,8935(646(4(())100(()) :100

33

Physicochemicalcharacterizationoflipidnanoparticles

Hydrodynamic diameter and ζ-potential of lipid nanoparticles/siRNA complexes were measured at 20°C by

DynamicLightScatteringusingaMalvernZetasizerNanoZS(Malvern,Worcestershire,UK),usingtheautomatic

algorithmmode.Forsizemeasurements,lipidnanoparticlesandsiRNAwerecomplexedtogether(260nMfinal

siRNA;200µLfinalvolume)in5%dextroseasdescribedabove.Complexeswerethendilutedwith800µLOpti-

MEM®, equilibrated for 20 minutes at 20°C and measured. Size measurements are reported using the Z-

Average value. ζ-potential measurements were realized at 20°C using the Smoluchowski model. Lipid

nanoparticlesandsiRNAarecomplexedtogether(260nMfinalsiRNA;200µLfinalvolume)in5%dextroseas

describedpreviously. Complexes are thendilutedwith800µLdextrose5%,equilibrated15minutes at 20°C

andmeasured.Experimentswererunintriplicateormore.

34

InvitrosiRNAtransfectionassays

TheRNAiinducedsilencingcapabilitiesofeachcationicswitchablelipidwasinvestigatedinaHeLa/GFPmodel.

Lipid nanoparticles (made by manual extrusion) and anti-GFP siRNA were complexed (N/P ratio of 4) as

describedabove(forafinalsiRNAconcentration/wellof1-60nM)andweredilutedinOpti-MEM®(250µL).This

reverse transfection media (300 µL) was put in the well (12-well plates), and 1 mL of cell suspension in

completeculturemedia(EMEM/FBS90:10)wasaddedforafinalconcentrationof40000cells/well.Cellswere

thenincubatedfor72hours.After incubation,cellswererinsedwithPBS,trypsinizedandsuspendedinFACS

buffer (95% PBS, 5% FBS, 1.0 mM EDTA) for immediate analysis on a FACSCaliburTM flow cytometer (BD

Biosciences,SanJose,CA,USA).GFPexpressionforeachreplicate(meanfluorescentintensity;FlowJosoftware

vX.0.7, Ashland, OR, USA) was calculated relative to the control samples that did not receive any siRNA

treatment. Negative control included cells that were treated with naked anti-GFP siRNA (60 nM). Positive

control included cells that were treatedwith Lipofectamine® RNAiMAX (Thermo Scientific) according to the

manufacturer’s reverse transfection protocol (1 µL reagent/well; 60 nM siRNA/well). All experiments were

realizedintriplicate.

The RNAi silencing capabilities of the formulations made by microfluidic mixing (aimed for in vivo siRNA

delivery) was assayed in HeLa/GFP (anti-GFP siRNA) and Huh-7 cells (anti-PCSK9 siRNA), using the same

protocol. Transfection efficiencywas assayed by either flow cytometry orwestern blotting7 after a 48 hour

(HeLa/GFP)or72hour(Huh-7)incubationperiod.

Forward transfection was also realized to make sure that transfection efficiency of CSL3 and CSL4 lipid

nanoparticles remains similar, regardlessof the chosen transfectionmethod.HeLa/GFP cellswere seeded in

12-wellplates(40000cells/well)andallowedtoattachovernight.Thenextday,cellswereincubatedinOpti-

MEM® for 4 hours with lipid nanoparticles (made by manual extrusion) or Lipofectamine® RNAiMAX (1 µL

reagent/well; 25nM siRNA/well).After incubation, cellswere rinsedwithOpti-MEM®and1mLof complete

culturemedia (EMEM/FBS90:10)wasadded.Cellswere then incubated for44hours.After incubation, cells

wererinsedwithPBS,trypsinizedandsuspendedinFACSbufferforimmediateanalysisviaflowcytometry.

35

Invitrocytotoxicityassay

Cytotoxicity of the lipid nanoparticles (made bymanual extrusion) on HeLa/GFP cells was assessed using a

resazurin-based cell viability assay (AlamarBlue®). The transfection procedure was strictly identical to that

presentedabove(N/Pratioof4,1-60nMfinalsiRNAconcentration/well),but theassaywascarefullyscaled

downfrom12-wellplatesto96-wellplates,accordingtothegrowthareaofthewells(3000cells/well,200µL

final volume/well). After 72 hours of incubation, cells were washed with PBS, and 200 µL of fresh culture

mediumwasaddedintothewells(EMEM/FBS90:10).20µLofafreshlypreparedresazurinsolution(440µMin

PBS)werethenadded,andcellswereincubatedfor2hours.Theabsorbanceofeachwellwasmeasuredat570

and600nmusingaSafiremicroplatereader(Tecan).Cellularviabilitywasnormalizedrelativetothenegative

control(cellstreatedwithdextrose5%).Experimentswereruninsixplicata.

36

IntracytoplasmicdeliveryofsiRNA

Live-cellfluorescencemicroscopywasusedtoimagetheintracytoplasmicdeliveryofsiRNA-Alexa647(Qiagen)

following transfection with the CSL3-based formulation (CSL3/DSPC/cholesterol/DSPE-PEG2000 50:10:37.5:2.5

mol% -madebymanual extrusion).HeLa cellswere routinely incubated24hoursbefore imaging in 35mm

poly-d-lysine coated glass dishes (MatTek Corporation, Ashland,MA,USA) at a density of 40 000 cells/dish.

siRNAwerecomplexedwithlipidnanoparticles(N/Pratioof4) in5%dextroseasdescribedpreviously(70µL

finalvolume).Complexeswere thendilutedwith346µLOpti-MEM®and1384µLofcompleteculturemedia

(Opti-MEM®/FBS90:10),forafinalsiRNAconcentrationof50nM.Cellswerethenincubated10minutes,1hour

or 2 hours with the lipid nanoparticles. After incubation, cells were rinsed four times with Opti-MEM®/FBS

90:10 andwere immediately imaged in a phenol red-free culturemedia (Opti-MEM®/FBS 90:10). Imaging of

HeLacellswasperformedusinganOlympusIX81fluorescentmicroscopeequippedwithaPlanApoN60X1.42

NA silicone objective (Olympus Canada Inc., Toronto, ON, Canada) and a 12 bits Retiga-2000R CCD Camera

(QImaging,Surrey,BC,Canada),usingMetaMorphAdvancedsoftware7.8.9(MolecularDevices,SanJose,CA,

USA). siRNA-Alexa647 was imaged using the Cy5 channel (λex/em 649/666). All fluorescence images were

carefullyexportedwithconstantscalingandrangeofgreylevel.Experimentwasrealizedintriplicate.

37

EndosomalentrapmentoftheCSL4-basedformulation

Live-cellfluorescencemicroscopywasusedtocomparetheintracellularfatefollowingendocytosisoftheCSL3-

based (pH-sensitive) and the CSL4-based (negative control) lipid nanoparticles formulations

(CSL/DSPC/cholesterol/DSPE-PEG2000 50:10:37.5:2.5 mol% - made by manual extrusion). HeLa cells were

routinelyincubated24hoursbeforeimagingin35mmpoly-d-lysinecoatedglassdishes(MatTekCorporation)

atadensityof40000cells/dish.siRNA-Alexa647(Qiagen)werecomplexedwithlipidnanoparticles(N/Pratio

of4) in5%dextroseasdescribedpreviously (70µL final volume).Complexeswere thendilutedwith346µL

Opti-MEM®and1384µLofcompleteculturemedia(Opti-MEM®/FBS90:10),forafinalsiRNAconcentrationof

50 nM. Cellswere then incubated 16 hourswith the lipid nanoparticles. After incubation, cellswere rinsed

twicewithOpti-MEM®/FBS90:10andwere stainedwithHoechst33342 (5min,2µg/mL, Sigma).Cellswere

rinsed twice with Opti-MEM®/FBS 90:10 and were immediately imaged in a phenol red-free culture media

(Opti-MEM®/FBS90:10).Alternatively,cellswereexposedto500µMchloroquineduring the lasthourof the

16-hour incubationperiod (CSL4-based formulation). ImagingofHeLacellswasperformedusinganOlympus

IX81 fluorescentmicroscope equippedwith a UPlanSApo 100X 1.40 NA silicone objective (Olympus Canada

Inc.) and a 12 bits Retiga-2000R CCD Camera (QImaging), using MetaMorph Advanced software 7.8.9

(MolecularDevices).Hoechst33342was imagedusingtheDAPIchannel(λex/em350/470)andsiRNA-Alexa647

was imaged using the Cy5 channel (λex/em 649/666). All fluorescence images were carefully exported with

constantscalingandrangeofgreylevel.Experimentwasrealizedintriplicate.

ComparisonofthecellularuptakeoftheCSL3andCSL4-basedformulationsoverthe16-hourincubationperiod

wasassayedbyflowcytometry.HeLacellswereseeded in12-wellplates(100000cells/well)andallowedto

attach overnight. The next day, lipid nanoparticles (25 µL in dextrose 5%) and siRNA-Alexa488 (25 µL in

dextrose 5%) were complexed in dextrose 5% (N/P ratio of 4; siRNA concentration 25 nM/well) and were

dilutedinOpti-MEM®(250µL)and1mLofcompleteculturemedia(EMEM/FBS90:10).Thesolutionwasthen

transferred onto the cells (1300 µL final volume/well) and the plate was incubated for 16 hours. After

incubation,cellswererinsedthreetimeswithEMEM/FBS90:10,oncewithPBS,trypsinizedandsuspendedin

FACS buffer (95% PBS, 5% FBS, 1.0 mM EDTA) for immediate analysis via flow cytometry. Experiment was


38

Lipid-mixingassayThefusogenicpotentialoftheCSL3/siRNAandCSL4/siRNAlipidnanoparticleswasevaluatedwithanoctadecyl

RhodamineB(R18) lipid-mixingassay.1,8R18wasincorporatedintothelipidnanoparticlesataself-quenched

concentrationof 6mol%.Unlabeled130nmPOPC vesicleswereprepared in 5%dextroseusing themanual

extrusionmethodpreviouslydescribed(9passagesthrougha200nmpolycarbonatemembrane).R18labeled

lipid nanoparticles (CSL/DSPC/cholesterol/DSPE-PEG2000/R18 50:10:31.5:2.5:6 mol% - made by manual

extrusion)were complexedwith anti-GFP siRNA as described above (N/P ratio of 4, in 5% dextrose). These

labeled lipidnanoparticlesweremixedwithunlabeledPOPCvesicles inabufferatpH7.4(5mMHEPESwith

ionicstrengthadjustedto150mMwithNaCl)oratpH5(50mMaceticbufferwithionicstrengthadjustedto

150mMwithNaCl),asdescribedbelow.

In3000µLofbuffercontainingunlabeledPOPCvesicles(100µMtotallipidcontentinthecuvette)wereadded

thelabeledlipidnanoparticles/siRNA(50µLvolume;10µMtotal lipidcontentinthecuvette).Theincreasing

fluorescence of R18 was monitored (λex/em 556/590, slits 5 nm) using a Hitachi F-2710 Spectrophotometer

equipped with a water circulated cell holder with stirring. All experiments were performed at 20°C under

mediumstirring.After15minutes,4µLofpureoctaethyleneglycolmonododecylether(Sigma)detergent(2.5

mMfinalconcentrationinthecuvette)wasaddedtoobtainthe100%dequenchedfluorescenceintensity.Raw

data were recorded continuously during the experiment. The percentage of membrane fusion activity at a

giventimetwasdefinedas:

%;8,-2(< ==< − =>

=?@@% − =>:100

whereIoistheinitialfluorescenceintensityobservedimmediatelyafteradditionoflabeledlipidnanoparticles;

I100%isthemaximalfluorescenceintensityvalueobtainedafteradditionofdetergentandItisthefluorescence

intensityvaluemeasuredatagiventimet.Experimentswererealizedintriplicate.

39

InhibitionofGFPknockdownwithBafilomycinA1

In this experiment, HeLa/GFP cells were transfected with the CSL3-based lipid nanoparticles formulation

(CSL3/DSPC/cholesterol/DSPE-PEG2000 50:10:37.5:2.5 mol% - made by manual extrusion) in the presence or

absenceofBafilomycinA1,aknowninhibitorofvacuolarH+ATPases.HeLa/GFPcellswereseeded in12-well

plates(40000cells/well)andallowedtoattachovernight.Thenextday,cellswererinsedwithOpti-MEM®and

pre-incubated 30minuteswith Bafilomycin A1 (600 nM/well, diluted in Opti-MEM®) before incubationwith

lipidnanoparticles.Lipidnanoparticlesandanti-GFPsiRNAwerecomplexed(N/Pratioof4)asdescribedabove

(for a final siRNAconcentrationof10and25nM/well). Theywere thendilutedwithOpti-MEM®, containing

BafilomycinA1fora finalconcentrationof600nM/well.ResidualDMSOintheassaywas0.06%v/v(treated

cellsandnegativecontrol).Cellswerethenincubatedfor4hourswiththelipidnanoparticles.Afterincubation,

cells were rinsed twice with Opti-MEM® and 1 mL of complete culture media (EMEM/FBS 90:10 – with or

without 600 nMBafilomycinA1)was added. Cellswere then incubated for 44 hours. After incubation, cells

were rinsed with PBS, trypsinized and suspended in FACS buffer (95% PBS, 5% FBS, 1.0 mM EDTA) for

immediate analysis via flow cytometry. GFP expression for each replicate (mean fluorescent intensity) was

calculatedrelativetothecontrolsamplesthatdidnotreceiveanysiRNAtreatment.Experimentswererealized

intriplicate.

ComparisonofthecellularuptakeoftheCSL3-basedformulationinthepresenceorabsenceofBafilomycinA1

over the4-hour incubationperiodwasassayedby flowcytometry.HeLa cellswere seeded in12-well plates

(100000cells/well)andallowedtoattachovernight.Thenextday,cellswererinsedwithOpti-MEM®andpre-

incubated30minuteswithBafilomycinA1 (600nM/well, diluted inOpti-MEM®)before incubationwith lipid

nanoparticles. Lipid nanoparticles (25 µL in dextrose 5%) and siRNA-Alexa488 (25 µL in dextrose 5%) were

complexedindextrose5%(N/Pratioof4;siRNAconcentration25nM/well)andweredilutedwithOpti-MEM®,

containingBafilomycinA1forafinalconcentrationof600nM/well.Thissolutionwasthentransferredontothe

cells (1300µLfinalvolume/well)andtheplatewas incubatedfor4hours.After incubation,cellswererinsed

threetimeswithEMEM/FBS90:10,onetimewithPBS,trypsinizedandsuspendedinFACSbuffer(95%PBS,5%

FBS,1.0mMEDTA)forimmediateanalysisviaflowcytometry.Experimentswererealizedintriplicate.

40

Uptakeinhibitionbyflowcytometry

HeLacellswereincubatedwithsiRNA-Alexa488packagedwiththeCSL3-basedlipidnanoparticlesformulation

(CSL3/DSPC/cholesterol/DSPE-PEG2000 50:10:37.5:2.5 mol% - made by manual extrusion) in the presence or

absence of endocytosis inhibitors. Inhibitors were used as follows: chlorpromazine (clathrin mediated

endocytosisinhibitor)10µg/mL9,10;genistein(caveolaemediatedendocytosisinhibitor)200µM11;Pitstop2TM

(clathrin and caveolaemediated endocytosis inhibitor) 20 µM12; EIPA (macropinocytosis inhibitor) 50 µM.13

Discrimination of the clathrin and caveolae uptake pathways was not intended, because of the rather

questionable specificity of the pharmacological endocytosis inhibitors between these two pathways.12,14,15

ResidualDMSOintheassaywas0.2%v/v(treatedcellsandnegativecontrol).

HeLacellswereseeded in12-wellplates (100000cells/well)andallowedtoattachovernight.Thenextday,

cellswererinsedwithOpti-MEM®andpre-incubated15minuteswithendocytosis inhibitors (diluted inOpti-

MEM®)beforeincubationwithlipidnanoparticles.LipidnanoparticlesandsiRNA-Alexa488werecomplexedin

dextrose5%(N/Pratioof4;siRNAconcentration25nM/well)andweredilutedinOpti-MEM®.Thesolutionwas

thentransferredontothecells(1300µLfinalvolume/well)andtheplatewasincubatedfor1or6hours.After

incubation,cellswererinsedthreetimeswithOpti-MEM®/FBS90:10,oncewithPBS,trypsinizedandsuspended

inFACSbuffer(95%PBS,5%FBS,1.0mMEDTA)forimmediateanalysisviaflowcytometry.Experimentswere


Uptakeinhibitionwasalsoconfirmedbylive-cellfluorescencemicroscopy.HeLacellswereroutinelyincubated

24hoursbefore imaging in35mmpoly-d-lysinecoatedglassdishes (MatTekCorporation)atadensityof40

000cells/dish.Thenextday,cellswererinsedwithOpti-MEM®andpre-incubated15minuteswithendocytosis

inhibitors(dilutedinOpti-MEM®)beforeincubationwithlipidnanoparticles.siRNA-Alexa647werecomplexed

with lipid nanoparticles (N/P ratio of 4) as described previously (70 µL final volume). Complexeswere then

dilutedwithOpti-MEM®forafinalsiRNAconcentrationof25nM/dish.Cellswerethenincubated1hourwith

thelipidnanoparticles.Afterincubation,cellswererinsedtwicewithOpti-MEM®/FBS90:10andwerestained

with Hoechst 33342 (5min, 2 µg/mL, Sigma). Cellswere rinsed twicewithOpti-MEM®/FBS 90:10 andwere

immediately imaged in a phenol red-free culturemedia (Opti-MEM®/FBS 90:10). Imaging of HeLa cells was

performedusinganOlympus IX81 fluorescentmicroscopeequippedwithaPlanApoN60X1.42NA silicone

41

objective (Olympus Canada Inc.) and a 12 bits Retiga-2000R CCD Camera (QImaging), using MetaMorph

Advanced software 7.8.9 (Molecular Devices). Hoechst 33342 was imaged using the DAPI channel (λex/em

350/470)andsiRNA-Alexa647wasimagedusingtheCy5channel(λex/em649/666).Allfluorescenceimageswere

carefullyexportedwithconstantscalingandrangeofgreylevel.Experimentswererealizedintriplicate.

42

Lipidnanoparticlepreparationbymicrofluidicmixing

Lipidnanoparticleswereprepared according topreviously reportedprocedures16,17, using aNanoassemblrTM

microfluidic instrument with herringbone rapid mixing features (Precision Nanosystems, Vancouver, BC,

Canada).Cationicswitchablelipids,DSPC,cholesterolandDMG-PEG2000weresolubilizedinethanolatamolar

ratioof50:10:37.5:2.5.siRNAwasdilutedin25mMsodiumacetatebufferpH4.0.ThesiRNA/totallipidmass

ratiowas~0.06,correspondingtoaN/Pratioof4.Thetwosolutionsweremixedataflowrateof12mL/min,

atasiRNAsolution/lipidsolutionratioof3:1.Totalvolumeofabatchistypically2mL(1.5mLsiRNAsolution

mixedwith0.5mLoflipidsolution).Nanoparticleswerethendialyzedagainstsaline(NaCl0.9%)usingPur-A-

LyzerTM Maxi dialysis tubes MWCO 12-14 kDa (Sigma). If needed, nanoparticles were concentrated using

Amicon Ultra-0.5 30K centrifugal filters device (EMDMillipore, Etobicoke, ON, Canada). Nanoparticles were

then sterile filteredusinga0.2µmpolyethersulfone13mmsyringe filter (Pall Corporation,Mississauga,ON,

Canada)andstoredat4°Cuntiluse.ThesiRNAencapsulationefficiencywasquantifiedusingaRibogreenRNA

quantitationassay(ThermoScientific).18NanoparticlesizewasmeasuredbyDynamicLightScatteringinsaline

(0.9%NaCl)at20°Caspreviouslydescribed.

ThefinalsiRNAconcentrationwasdeterminedviaion-pairingreversedphaseliquidchromatography,according

toapreviouslyreportedprocedure.19siRNAwereanalyzedusingaWatersXTerra®MSC18column(2.1x50mm,

2.5µmparticlesize)at60°ConanAgilent1100HPLCsystem,withUVdetectionat260nm.MobilephaseA

was95%0.1Mtriethylamineammoniumacetate(TEAA)pH7.0and5%acetonitrile.MobilephaseBwas80%

0.1MTEAApH7.0and20%acetonitrile.Thegradientchangesfrom0%to70%mobilephaseBfor12minwith

aflowrateof0.4mL/min.Injectionvolumewas6µL.CalibrationcurvesofsiRNAduplexusuallyrangesfrom20

to100µg/mL.Beforeanalysis,analiquotofthelipidnanoparticleformulationwasdilutedwith200mMTEAA

pH7.0+2%(v/v)C12E8detergenttoliberateencapsulatedsiRNA.

43

Biodistributionandex-vivoimaginginmice

AllproceduresusedinanimalstudieswereapprovedbytheComitédeDéontologiede l’Expérimentationsur

lesAnimaux (CDEA–AnimalCareandEthicalCommittee)of theUniversityofMontrealandwereconsistent

with local, state and federal regulations. Mice were maintained at the animal facilities of the Institute for

Research in Immunology and Cancer (IRIC) at theUniversity ofMontreal, andwere housed under standard

conditions. Food andwaterwere providedad libitum. Animalswere acclimated to the animal facility for at

leastoneweekbeforeexperiments.Priortoinjection,lipidnanoparticlesformulationsweredilutedwithsaline

(NaCl0.9%)atsiRNAconcentrationssuchthateachmousewasadministeredadoseof10µL/gbody-weight.

8-week-oldmaleCD-1mice(CharlesRiver,Saint-Constant,QC,Canada)wereinjectedintravenouslyviathetail

veinwithCSL3-basedlipidnanoparticles(CSL3/DSPC/cholesterol/DMG-PEG200050:10:37.5:2.5mol%-madeby

microfluidicmixing)formulatedwithsiRNAlabeledwithCy5onthesensestrand,unformulatedfreesiRNA-Cy5

or saline at a siRNA dose of 1.5mg/kg. 4-hour post injection,micewere euthanized by CO2 inhalation and

organs(brain,lungs,heart,liver,spleen,kidneys)wereimmediatelyharvestedandimagedusingaOptixMX3

optical imaging system (AdvancedResearchTechnologies,Montreal,QC,Canada).Datawasprocessedusing

the OptiView® software from Advanced Research Technologies. Normalized photon counts (fluorescence

intensity)wasnormalizedbywetmassoforgans.Experimentswererealizedintriplicate.

44

Serumstabilitystudy

This studywasconductedaspreviouslydescribedbyZhouetal.20 Inorder toestimate the stabilityofCSL3-

based LNP (CSL3/DSPC/cholesterol/DMG-PEG2000 50:10:37.5:2.5 mol% - made by microfluidic mixing) in the

presence of plasma, DLSmeasurements of LNP/siRNA incubated into PBS, PBS/FBS 90:10 or PBS/FBS 50:50

(v/v)wererealizedusingaMalvernZetasizerNanoZS(thermostatedat37°C,automaticalgorithmmode).For

the storage stability study in PBS, LNP were kept at +4°C between each measurement. Experiments were


45

InvivoFactorVIIsilencinginmice

Prior to injection, lipidnanoparticles formulations (CSL/DSPC/cholesterol/DMG-PEG200050:10:37.5:2.5mol%-

made by microfluidic mixing) were diluted with saline (NaCl 0.9%) at siRNA concentrations such that each

mousewasadministeredadoseof10µL/gbody-weight.6to8-weeksoldfemaleC57BL/6mice(CharlesRiver,

Saint-Constant,QC, Canada) received tail vein intravenous injection of saline (negative control, n=6) or LNP

containing anti-Factor VII siRNA (n=4). After 48h, mice were euthanized and blood was collected via

intracardiacsamplingoncitratetubes.Serumwasseparatedfromwholebloodusingserumseparationtubes

(BectonDickinson,FranklinLakes,NJ,USA)andresidualserumFVIIlevelsweredeterminedusingtheBiophen

VIIchromogenicassay(Aniara,WestChester,OH,USA)accordingtomanufacturer’sprotocol.Astandardcurve

was constructed using samples from saline-injected mice (pooled plasma, n=6) and relative Factor VII

expressionwasdeterminedbycomparingtreatedgroupstothestandardcurve.

46

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article nanoscale V21 noyellow - Royal Society of · PDF file3 Table of content Materials, general methods and cell culture 21 Synthesis of the pH-sensitive cationic switchable lipids

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