enzyme-responsive polyion complex (pic) nanoparticles for the … · 2016-03-16 · antimicrobial...

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Enzyme-responsivepolyioncomplex(PIC)nanoparticlesforthetargeteddeliveryofantimicrobialpolymersIgnacioInsua,aEvangelosLiamas,bZhenyuZhang,bAnnaF.A.Peacock,aAnneMarieKrachlerc,dandFranciscoFernandez-Trilloa,d*aSchoolofChemistry,bSchoolofChemicalEngineering,cSchoolofBiosciencesanddInstituteofMicrobiologyandInfec-tion,UniversityofBirmingham,Edgbaston,B152TTBirmingham,UnitedKingdom.E-mail:[email protected]

1.MATERIALS 1

2.INSTRUMENTATION 2

3.SYNTHESISANDCHARACTERISATIONOFPEPTIDES 2

3.1.LOADINGOFFMOC-L-CYS(TRT)-OHON2-CHLOROTRITYLCHLORIDERESIN 23.2.SOLID-PHASESYNTHESISOFPEPTIDES 23.3.CHARACTERISATIONOFPEPTIDES 3

4.ENZYMATICDEGRADATIONOFPEPTIDES 6

4.1.SYNTHESISOFSUCCINYLCASEIN(SC) 6

5.ANTIMICROBIALACTIVITYOFCOMMERCIALPEIS 7

6.CHARACTERISATIONOFPICNANOPARTICLES 8

6.1.CROSS-LINKINGKINETICSOFPICNANOPARTICLES 8

7.PHYSIOLOGICALSTABILITYOFPICNANOPARTICLES 9

8.STABILITYINTHEPRESENCEOFDIVALENTCATIONS–TREATMENTWITHCaCl2 11

9.ENZYMATICDEGRADATIONOFPICNANOPARTICLES 12

10.ANTIMICROBIALACTIVITYOFPICNANOPARTICLES 13

11.SUPPLEMENTARYREFERENCES 14

1. MaterialsFmoc-protected-L-amino acidswere purchased fromMerckMillipore. Dimethylformamide (DMF), piperidine 20%v/v inDMF, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and linear poly(ethylene imine) 2.5 KDa averagemolecularweight (L-PEI2.5) were purchased from Sigma-Aldrich®. H2N-L-Glu(OtBu)-2-chlorotrityl resin (0.58 mmol/g) wasbought from AGTC Bio Products Ltd. 2-chlorotrityl chloride resin (1.49 mmol/g) was obtained from Iris BiotechGmbh. Linearpoly(ethylene imine) 25KDaaveragemolecularweight (L-PEI25), branchedpoly(ethylene imine) 1.2KDa average molecular weight (B-PEI1.2), N,N-diisopropylethylamine (DIPEA), triisopropylsilane (TIPS), 1,2-ethanedithiol (EDT), trifluoroacetic acid (TFA), casein from bovine milk (technical grade) and succinic anhydridewere bought from Alfa Aesar®. N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

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





(HBTU)waspurchased fromCarbosynthLtd.Spectra/Por®6dialysismembranes (molecularweightcutoff1KDa)were purchased from Spectrum® Laboratories. Penta(ethylene imine) (L-PEI5n)was purchased fromAcrosOrgan-icsTM.AllotherchemicalswerepurchasedfromFisherScientificUKLtd.andwereusedwithoutfurtherpurification.

2. InstrumentationNMRdatawasacquiredonaBrukerAvanceIIIoperatingat400MHzandfittedwitha5mmDULprobe(1H/13C).MSspectrawereobtainedon a Xevo®G2-XS ToF (Waters) fromelectrospray ionisation (ESI) and time-of-flight (TOF)measurementinpositiveionmode.High-resolutionMSdatawascalculatedbycomparisonwithleucine-enkephalinas internal standard. Reverse phase (RP) HPLC analysis was run through a Kinetex® C18-EVO column (Phenom-enex®):5µm,100Å,250x4.60mm.Agradientfrom3to20%of(CH3CN+0.05%TFA) in(H2O+0.05%TFA)wasused at 1mL/min. The columnwasmaintained at 35°C andUV-VIS detectionwas set at 210nm.UV-VIS spectrawereacquiredonaCary5000NIR (Agilent) inpolystyrenecuvettesof1mm lightpaths.Dynamic light scattering(DLS)andζ-potentialmeasurementswerecarriedoutinaZetasizerNanoZS(MalvernInstrumentsLtd)stabilisedat37°C.DLSwasreadat173°(backscattering)for60secondsintriplicateandζ-potentialswererecorded30timesat140V.

3. Synthesisandcharacterisationofpeptides

3.1. LoadingofFmoc-L-Cys(Trt)-OHon2-chlorotritylchlorideresin

AsolutionofFmoc-L-Cys(Trt)-OH(1.10g,1.9mmol)in10mLofDCM:anhydrousDMF(9:1)waspurgedwithargonfor10min.Then,thesolutionwasaddedunderinertconditionsintoasealedflaskwithcommercialpreswollen2-chlorotritylchlorideresin(1.00g,1.5mmol),andDIPEA(1.3mL,7.5mmol)wasaddedtothemixture.Thereactionwasleftunderstirringatroomtemperaturefor2hours.Afterthistime,theresinwasfilteredandwashedwith10mLofDCM:MeOH:DIPEA(17:2:1)3times.Finally,theresinwaswashedwith10mLofDCM(x3)and10mLofEt2O(x3) anddriedunder vacuum.Amino acid loadingwas calculated following the protocol describedby Kay et al.,1

givingafinalvalueof0.44mmol/gofresin.

3.2. Solid-phasesynthesisofpeptidesForpeptideswithoutcysteine(P1,P2andP3),commercialH2N-L-Glu(OtBu)-2-chlorotritylresin(250-400mg,0.14-0.23mmol)was swollen in 4mLofDMF for 30minutes. Then, solutionsof Fmoc-L-amino acid (3 eq),N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate(HBTU)(2.8eq)andDIPEA(2.8eq)inDMFwereaddedtoafinalvolumeof4mL.Thereactionmixturewasshakenfor1houratroomtemperature,afterwhichanegativechloranil test2 indicatedthereactionhadgonetocompletion.N-terminalFmocprotectinggroupwasre-movedwith4mLofpiperidineinDMF20%v/vduring10minutes.ApositivechloraniltestconfirmedtheremovaloftheFmocgroup,andthepreviousstepswererepeatedtocoupleallaminoacidsinthesequence.Followingcou-plingofthelastaminoacid,theFmocgroupwasremovedand,ifnecessary,theterminalamineinthepeptidewascapped with 4 mL of Ac2O:DIPEA:DMF (1:1:3) for 1 hour at room temperature. Then, the resin was thoroughly

Cl

Cl

HO

OHN O

OS

+ O

Cl

OHN

S

O

O





washedwithEt2Oandthepeptidewascleavedfromtheresinwith4mLofaqueousTFA(95%)for1hour.Thesolu-tionobtainedwas concentratedunder argon andprecipitated in chilled Et2O. Finally, this suspensionwas centri-fugedandthepelletswerefreeze-driedfromacidicwater.ForpeptideP1SH(containingcysteine),Fmoc-L-Cys(Trt)-OHloaded2-chlorotritylchlorideresin(1.00g,0.44mmol),preparedasindicatedin3.1.,wasdeprotectedtwicewith10mLofpiperidineinDMF20%v/vbeforecouplingsub-sequentaminoacids.TheelongationandcappingofthepeptidewasperformedasindicatedaboveforP1-P3,andthepeptidewascleaved fromtheresinwith10mLofTFA (80%),TIPS (8%)andEDT (12%) for2hours toprotectthiolsfromsidereactions.ThissolutionwasconcentratedunderargonandprecipitatedinchilledEt2O:hexane(1:4).Finally,thissuspensionwascentrifugedandthepelletswerefreeze-driedfromacidicwater.Allpeptideswereiso-latedaswhitesolids,regardlessofthesyntheticmethod.PuritywasdeterminedbyHPLC.

TableS1Sequencesandstructuresofthepeptidespreparedinthiswork.

Peptide Sequence Structure

P1 Ac-EGLAE-OH

P1SH Ac-CEGLAEC-OH

P2 Ac-EAAAAE-OH

P3 H2N-LAE-OH

3.3. CharacterisationofpeptidesP1, Ac-E-GLA-E-OH (83.8 mg, 96% yield) 1H-NMR (400 MHz, DMSO-d6): δ 0.84(dd, J=15.1, 6.5 Hz, 6H, Hδ-Leu);1.21(d, J=7.0Hz,3H,Hβ-Ala);1.36-1.49(m,2H,Hβ-Leu);1.51-1.61(m,1H,Hγ-Leu);1.67-2.00(m,4H,Hβ-Glu);1.85(s,3H,Ac);2.20-2.33(m,4H,HΥ-Glu);3.68(d,J=5.8Hz,2H,Hα-Gly);4.15-4.34(m,4H,Hα);7.83(d,J=8.2Hz,1H,NHCO);7.96(d,J=7.8Hz,1H,NHCO);8.04(d,J=7.4Hz,1H,NHCO);8.11(d,J=7.4Hz,1H,NHCO);8.21(t,J=5.8Hz,1H,NHCO)ppm.13C-NMR(400MHz,DMSO-d6):δ17.9(Cβ-Ala);21.6(Cδ-Leu);22.5(Ac);23.2(Cδ-Leu);24.1(Cγ-Leu);26.4(Cβ-Glu);27.1(Cβ-Glu); 30.0(Cγ-Glu); 30.2(Cγ-Glu); 40.8(Cβ-Leu); 42.0(Cα-Gly); 48.0(Cα); 50.8(Cα); 51.1(Cα); 52.3(Cα);168.6(NHCO); 169.8(Ac); 171.6(NHCO); 171.8(NHCO); 172.2(NHCO); 173.1(C-term); 173.8(Cδ-Glu); 174.0(Cδ-Glu)ppm.MS(ESI-TOF,+eV):m/z614.2[M+Na+CH3OH]+;604.2[M-H+2Na]+;582.2[M+Na]+.HR-MS(ESI-TOF,+eV):m/z582.2387(calculatedfor[M+Na]+)582.2385(found).PuritybyHPLC=88%.P1SH, Ac-C-E-GLA-E-C-OH (214.3mg, 64% yield). 1H-NMR (400MHz,DMSO-d6): δ 0.84(dd, J=14.8, 6.5Hz, 6H,Hδ-Leu);1.20(d,J=7.1Hz,3H,Hβ-Ala);1.43(t,J=7.0Hz,2H,Hβ-Leu);1.50-1.63(m,1H,Hγ-Leu);1.67-1.83(m,2H,Hβ-Glu);1.84-1.98(m,2H,Hβ-Glu);1.87(s,3H,Ac);2.18-2.29(m,4H,HΥ-Glu);2.34(t, J=8.5Hz,1H, SH);2.44(t, J=8.5Hz,1H,SH);2.62-2.90(m,4H,Hβ-Cys);3.70(dd, J=11.2,5.7Hz,2H,Hα-Gly);4.19-4.41(m,6H,Hα);7.87-7.92(m,2H,NHCO);8.07-8.19(m,5H,NHCO)ppm. 13C-NMR (400MHz,DMSO-d6): δ17.7(Cβ-Ala); 21.6(Hδ-Leu); 22.6(Ac); 23.1(Hδ-Leu);

HN

O

NH

OHN

O

NH

OHN

O

OH

O

OHO OHO

NH

HN

O

O

NH

OHN

O

NH

OHN

O

NH

OSH

OH

O

SH

OHO OHO

NH

HN

O

O

NH

OHN

O

NH

OHN

O

OH

O

OHO

OHO

H2NNH

OHN

O

OH

O

OHO





24.1(HΥ-Leu);25.4(Cβ-Cys);26.0(Cβ-Cys);27.1(Cβ-Glu);27.5(Cβ-Glu);29.9(Cγ-Glu);30.0(Cγ-Glu);41.0(Cβ-Leu);41.9(Cα-Gly);48.2(Cα);50.8(Cα);51.6(Cα);52.2(Cα);54.3(Cα);55.1(Cα);168.4(NHCO);169.7(Ac);170.2(NHCO);171.1(NHCO);171.3(NHCO); 171.4(NHCO); 171.8(NHCO); 172.0(C-term); 174.0(Cδ-Glu) ppm.MS (ESI-TOF, +eV): m/z 810.2 [M-H+2Na]+,788.3[M+Na]+.HR-MS(ESI-TOF,+eV):m/z788.2571(calculatedfor[M+Na]+)788.2568(found).PuritybyHPLC=92%.P2,Ac-E-AAAA-E-OH(118.8mg,97%yield)1H-NMR(400MHz,DMSO-d6):δ1.18-1.21(m,12H,Hβ-Ala);1.64-2.00(m,4H,Hβ-Glu);1.84(s,3H,Ac);2.17-2.33(m,4H,HΥ-Glu);4.16-4.30(m,6H,Hα); 7.91(d, J=7.4Hz,2H,NHCO);7.99(d,J=7.3Hz,1H,NHCO);8.03(d,J=8.1Hz,1H,NHCO);8.05(d,J=7.7Hz,2H,NHCO)ppm.13C-NMR(400MHz,DMSO-d6):δ 17.9(Cβ-Ala); 18.1(Cβ-Ala); 18.1(Cβ-Ala); 22.5(Ac); 26.4(Cβ-Glu); 27.4(Cβ-Glu); 30.0(Cγ-Glu); 30.2(Cγ-Glu); 47.8(Cα);48.0(Cα);48.1(Cα);48.1(Cα);51.1(Cα);51.8(Cα);169.4(Ac);171.1(NHCO);171.6(NHCO);171.8(NHCO);171.9(NHCO);172.2(C-term); 173.1(NHCO); 173.7(Cδ-Glu); 174.0(Cδ-Glu) ppm.MS (ESI-TOF, +eV):m/z 647.2 [M-H+2Na]+; 625.3[M+Na]+.HR-MS(ESI-TOF,+eV):m/z625.2445(calculatedfor[M+Na]+)625.2444(found).PuritybyHPLC=98%.P3,H2N-LA-E-OH (50.4mg,66%yield)1H-NMR (400MHz,DMSO-d6):δ0.88(dd,J=8.8,6.4Hz,6H,Hδ-Leu);1.24(d,J=7.0Hz,3H,Hβ-Ala);1.45-1.59(m,2H,Hβ-Leu);1.61-1.69(m,1H,Hγ-Leu);1.71-1.81(m,1H,Hβ-Glu);1.91-2.00(m,1H,Hβ-Glu);2.19-2.32(m,2H,HΥ-Glu);3.77(t,J=6.6Hz,1H,Hα-Leu);4.16-4.21(m,1H,Hα-Glu);4.38(p,J=7.1Hz,1H,Hα-Ala);8.21(d,J=7.8Hz,1H,NHCO);8.69(d,J=7.4Hz,1H,NHCO)ppm.13C-NMR(400MHz,DMSO-d6):δ18.1(Cβ-Ala);22.0(Hδ-Leu);22.7(Hδ-Leu);23.6(Cγ-Leu);26.5(Cβ-Glu);30.1(Cγ-Glu);40.3(Cβ-Leu);48.2(Cα-Ala);50.8(Cα-Leu);51.3(Cα-Glu);168.3(NHCO);171.7(NHCO);173.1(C-term);173.9(Cδ-Glu)ppm.MS (ESI-TOF,+eV):m/z332.2[M+H]+.HR-MS(ESI-TOF,+eV):m/z332.1822(calculatedfor[M+H]+)332.1824(found).PuritybyHPLC=99%.

Fig.S11H(left)and13C(right)NMR(400MHz,DMSO-d6)spectraofP1(Ac-E-GLA-E-OH).





Fig.S21H(left)and13C(right)NMR(400MHz,DMSO-d6)spectraofP1SH(Ac-C-E-GLA-E-C-OH).

Fig.S31H(left)and13C(right)NMR(400MHz,DMSO-d6)spectraofP2(Ac-E-AAAA-E-OH).

Fig.S41H(left)and13C(right)NMR(400MHz,DMSO-d6)spectraofP3(H2N-LA-E-OH).





Fig.S5ESI-TOFmassspectraofP1(Ac-E-GLA-E-OH),P1SH(Ac-C-E-GLA-E-C-OH),P2(Ac-E-AAAA-E-OH)andP3(H2N-LA-E-OH).

Fig.S6RP-HPLCchromatogramsofP1(Ac-E-GLA-E-OH),P1SH(Ac-C-E-GLA-E-C-OH),P2(Ac-E-AAAA-E-OH)andP3(H2N-LA-E-OH).

4. Enzymaticdegradationofpeptides

4.1. Synthesisofsuccinylcasein(SC)Amodified procedure from the literaturewas used for the preparation of SC:3 commercial casein (500mg, 0.02mmol)washeatedat60°Cwithsuccinicanhydride(400mg,4.00mmol)in100mLofNaHCO3bufferatpH8.0.ThepHof thereactionwasmaintainedat8.0byadditionofNaOH1M.After1hour, thereactionmixturewas left toreachroomtemperatureanddialysedagainstdeionisedwaterfor2days.Then,thesuspensionwasfreeze-driedtogiveawhitesolid(481.8mg).SCwasusedasacontroltomonitorenzymaticactivitybecauseSCisdegradedbybothproteases.Foreachexperi-ment,degradationofSCwasmonitoredtoensurethatenzymaticactivityforLasBandHLEwasnotlostuponstor-age.Ingeneral,weobservedahigherdegradationofSCinthepresenceofHLE(Fig.S7).ThisincreaseinenzymaticdegradationbyHLEcanbeduetoahighercontentofHLEcleavablesitesinSC.Thus,wepreparedamodeldegrada-tion peptide (H2N-LA-E-OH,P3) that corresponds to the product obtainedupon enzymatic hydrolysis ofP1.4 Thispeptidehasaterminalamineandisnotcleavedbyanyoftheelastases.Thus,fluorescenceintensitycanbenormal-isedtothatobservedinthepresenceofthispeptide,whichcorrespondsto100%degradation.





Fig.S7Emissionintensity(λexc355nm,λem460nm)offluorescamineconjugatesforNa2B4O7buffer(blank),succinylcasein(SC)asacontrolforenzymaticactivity,LasBresponsiveanionicpeptides(Ac-E-GLA-E-OH,P1;Ac-E-AAAA-E-OH,P2)anddeg-radation peptide (H2N-LA-E-OH, P3) as a control to normalise fluorescence intensity. All substrateswere evaluated in theabsenceandpresenceofLasBorHLE.Incubationtime:4hours.n=3.

5. AntimicrobialactivityofcommercialPEIs1mLaliquotsofaP.aeruginosaPAO1VcultureinLBbroth(OD600=1.0)werecentrifugedandresuspendedinthesamevolumeofaPEIsolutionpreparedinDMEM.5differentPEIsweretested:3linear(25KDa,2.5KDaandpen-tamer) and 2 branched (25 and 1.2 KDa) at 3 different concentrations. Bacteria were also resuspended in pureDMEMand70%v/vaqueous2-propanolasliveanddeadcontrols,respectively.Allsampleswereincubatedatroomtemperaturefortwentyhours.Afterthistime,300µLofeachsamplewerereacted inthedarkwith1µLofa1:1mixtureofBacLightTMprobesfor10minutes.Then,sampleswereanalysedbyFACSsettinggatesingreenversusredemissiondotplotsfromtheliveanddeadcontrols.Forsomeofthehigherconcentrations,aggregation,biofilmsorbacterialdebriswereobservedintheculturewellbeforeFACSanalysis.

0 50,000 100,000 150,000 200,000 250,000

Blank

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SC

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SC+HLE

P1

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P1+HLE

P2

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P3

P3+LasB

P3+HLE

Emission intensity





Fig.S8NormalisedpopulationofP.aeruginosaPAO1Vpresentedasthepercentageofred(dead)cells.n=1.

6. CharacterisationofPICnanoparticlesTableS2Hydrodynamicdiameter(DH)andζ-potentialofPICnanoparticlespreparedatdifferentN:COOHratios.SDindicatesthedispersioninDHorζoftheonlysizeorchargepopulationfittedbythesoftware.

N:COOHRATIO DH±SD(nm) ζ±SD(mV) Notes

1:2.0 - - NoPICparticlesformed



1:0.8 582±79 6.7±4.7 -

1:0.7 487±67 9.8±3.2 -

1:0.6 431±59 11.3±4.5 -

1:0.5 379±68 13.4±4.7 -

1:0.4 230±56 17.2±7.1 -

1:0.3 111±35 18.9±6.4 -


6.1. Cross-linkingkineticsofPICnanoparticlesEllman'sassaywasusedtoquantifybyUV-VISspectroscopytheamountofaccessiblesulfhydrylgroupspresentinthesample.PICnanoparticleswerepreparedata1:0.3N:COOHratioasdescribedinthemaintext.300µLofsam-pleweretakenatdifferenttimepointsandmixedwith300µLof1mM5,5'-dithiobis(2-nitrobenzoicacid) (DTNB)solutionindegassed100mMphosphatebufferatpH7.27containingwith1mMEDTA.Absorbancewasmeasuredat412nm.ThiolconcentrationinasolutionofP1SHatthesameconcentrationwasusedasacontrol.

L-PEI5n L-PEI2.5 L-PEI25 B-PEI1.2 B-PEI25

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20

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% D

ead

cells

0.1 mg/mL0.01 mg/mL0.001 mg/mL

!

!

! = biofilm formation





Fig.S9Amountofaccessiblethiols,asmonitoredusingEllman'sassay,followingincubationofP1SH(○)orPICnanoparticles(●)preparedata1:0.3N:COOHratioinphosphate/EDTAbuffer(pH7.27)understirringandopentoair.n=1.

7. PhysiologicalstabilityofPICnanoparticles1mLofaPICnanoparticles (TableS2),preparedasdescribed in themain text,wasdilutedwith182µLofa1MNaCl solution inwater togivea finalNaCl concentrationof154mM(physiologicalosmolarity).5All sampleswereincubatedat37°CandanalysedbyDLSovertimeforupto4hours.Autocorrelationfunction(ACF),intensitydistri-butionsanddetectioncountsforthesesampleswerecomparedtothatofacontrolintheabsenceofNaCl(Fig.S10-Fig.S12).

0 2 4 6 8 100

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aini

ng fr

ee th

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P1SH





Fig.S10Representativeautocorrelationfunction†(ACF)curvesforP1SHPICnanoparticlespreparedusingsixdifferentN:COOHratios(TableS2)intheabsence(control)andpresenceof154mMNaClat37°Covertime(1-4hours).

†BecauseofthedispersioninDLSmeasurementsobservedforunstableparticles(e.g.1:0.6N:COOHratioat3hours)onlyrepresentativeplotsareshown.3technicalreplicateswererecordedforeachsample.

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Fig.S11Representativeintensitydistributions†ofsizesforthefourmoststableP1SHPICnanoparticlesanalysed(TableS2andFig.S10)intheabsence(control)andpresenceof154mMNaClat37°Covertime(1-4hours).

Fig.S12NormaliseddetectioncountsforP1SHPICnanoparticlespreparedusingsixdifferentN:COOHratios(TableS2)intheabsence(0h)andpresenceof154mMNaClat37°Covertime(1-4hours).DatawasnormalisedtothesizeoftheparticlesintheabsenceofNaCl(100%).Valuesplottedaretheaverageof3technicalreplicates.

8. Stabilityinthepresenceofdivalentcations–TreatmentwithCaCl2To500µLofPICparticles(1:0.6inN:COOH),preparedasdescribedinthemaintext,a1MsolutionofCaCl2inwaterwasaddedtogiveafinalconcentrationof10,50and100mMinCa2+.ThesampleswereanalysedbyDLSimmedi-atelyafteradditionofCaCl2.

0 1 2 3 40

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nts

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1 : 0.81 : 0.71 : 0.6

1 : 0.51 : 0.41 : 0.3





Fig.S13 Representative DLSautocorrelationfunction(ACF)curves(left)andintensitydistributionsofsizes(right)ofPICpar-ticles(1:0.6inN:COOH)intheabsence(control)andpresenceofincreasingconcentrationsofCaCl2.

9. EnzymaticdegradationofPICnanoparticles

Fig.S14RepresentativeintensitydistributionsofsizesforP1SHPICnanoparticlespreparedata1:0.3N:COOHratioincubatedwithNa2B4O7buffer(left),HLE(middle)andLasB(right)overtime.DistributionforLasBbroadensuponincreasingincubationtime(arrow).Valuesplottedaretheaverageof3samplereplicates,eachmeasured3times.n=9.

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PIC (1:0.3) control0 hours1 hour2 hours3 hours4 hours6 hours

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Fig.S15Emissionintensity(λexc355nm,λem460nm)offluorescamineconjugatesforenzymes,succinylcasein(SC)asacon-trolforenzymaticactivityandP1SHPICnanoparticlespreparedata1:0.3N:COOHratio.AllsubstrateswereevaluatedintheabsenceandpresenceofLasBorHLEandincubatedfor4hours.**p<0.01betweenPICandPIC+LasB.n=3.

10. AntimicrobialactivityofPICnanoparticles

Fig.S16RawFACSdataforP.aeruginosawildtype(PAO1V)andΔlasABmutantintheabsence(livecontrol,whitebars)andpresence ofP1SH PIC nanoparticles (1:0.3N:COOH ratio, grey bars), B-PEI25 (black bars) and 70% v/v aqueous 2-propanol(deadcontrol,dottedbars).Populationpresentedasthepercentageofred(dead)cells.Ineachgroupoffour,eachbarrep-resentsatimepoint(1,2,3and4hoursfromlefttoright).ToxicityofB-PEI25afteronehourwasnotstatisticallydifferentfromthelivecontrolandthusthattimepointhasnotbeenincludedinFig.5.n=3.

Blank LasB HLE SC SC+LasB SC+HLE PIC PIC+LasB PIC+HLE0

10,000

20,000

30,000

40,000

160,000

190,000

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50

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Live Dead PIC (1:0.3) B-PEI25

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Fig.S17RelativeantimicrobialactivityforP.aeruginosawildtype(PAO1V)andΔlasABinthepresenceofP1SHPICnanoparti-cles(1:0.3N:COOHratio,greybars)andB-PEI25(blackbars).RelativeantimicrobialactivitywascalculatednormalisingdatafromFig.S16tolivecontrol(0%ofantimicrobialactivity)anddeadcontrol(100%ofantimicrobialactivity)ateachtimepoint.n=3.

11. Supplementaryreferences1 C.Kay,O.E.Lorthioir,N.J.Parr,M.Congreve,S.C.McKeown,J.J.ScicinskiandS.V.Ley,Biotechnol.Bioeng.,2001,

71,110.2 T.Vojkovsky,Pept.Res.,1995,8,236.3 T.Hatakeyama,H.KohzakiandN.Yamasaki,Anal.Biochem.,1992,204,181.4 K.MoriharaandH.Tsuzuki,Arch.Biochem.Biophys.,1971,146,291–296.5 E.KesslerandD.E.Ohman,inHandbookofProteolyticEnzymes-(ThirdEdition)-ScienceDirect,eds.N.D.Rawlings

andG.Salvesen,AcademicPress,London,3rdedn.2013,vol.1,pp.582–592.

PAO1V + PIC (1:0.3) PAO1V + B-PEI25 ΔlasAB + PIC (1:0.3) ΔlasAB + B-PEI25

0

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1 h2 h3 h4 h

enzyme-responsive polyion complex (pic) nanoparticles for the … · 2016-03-16 · antimicrobial...

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