The aim of this study was to develop hepatocyte-targeting non-viral polymeric nono-carriers for gene delivery. Chitosan was selected as the main polymer. An asialoglycoprotein receptor recognized sugar, galactose, was introduced.
Trang 1jo u r n al h om ep a g e :w w w e l s e v i e r c o m / l o c a t e / c a r b p o l
Wen Jen Lina,b,∗, Wan Yi Hsua
a r t i c l e i n f o
Keywords:
Chitosan
Galactose
a b s t r a c t The aimof this study was todevelop hepatocyte-targeting non-viralpolymeric nono-carriers for genedelivery Chitosanwas selected asthe main polymer An asialoglycoproteinreceptor recog-nized sugar,galactose, was introduced Themethoxypoly(ethylene glycol)(mPEG)or shortchain poly(ethyleneglycol) diacid(PEGd) was further graftedonto galactosylatedchitosan All polyplex possessed positivecharge character.The compactionof DNAbygrafted chitosanwas in orderof chitosan-galactose-mPEG>chitosan-galactose-PEGd>chitosan-galactosewherethe chitosan-galactose-mPEG and pDNA formed the most stable polyplex The polyplex prominently enhanced DNA cellulartransfectionascomparedtonakedDNAinHepG2cellsinorderofchitosan-galactose/pDNA (11.6±0.6–33.0±4.4%)>chitosan-galactose-PEGd/pDNA (12.7±2.5–15.5±3.0%)> chitosan-galactose-mPEG/pDNA(9.0±1.1–12.9±2.4%)
©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND
license(http://creativecommons.org/licenses/by-nc-nd/3.0/)
1 Introduction
Chitosanisarelativelylowtoxic,biocompatible,and
biodegrad-ablepolysaccharidewithimmunological,antibacterialand
wound-healing activities Several strategies have been adopted for
chemical modification of chitosan through C2-amino group or
C6-hydroxylgroupusingdifferentsubstitutes(Gaoet al.,2009;
Gorochovceva &Makus, 2004; Laurentin &Edwards, 2003; Lin
& Chen, 2007; Liu et al., 2009; Park et al., 2003; Sajomsang,
Tantayanon, Tangpasuthadol, & Daly, 2009) The modified
chi-tosanisappliedfordrugdelivery,tissue engineering,andother
biomedicalapplications(Alves&Mano,2008;D’Amelioetal.,2013;
Muzzarelli,2010).ThefreeC2-aminogroupofchitosanisfeasible
tocomplexwithnegativelychargedDNAasagenedelivery
car-rier.Poly(ethyleneglycol)(PEG)ispopularlyusedinpharmaceutics
duetoitshydrophiliccharacter,highsolubility,lowcytotoxicity
andgoodbiocompatibility.Itwasreportedthat PEGcanreduce
proteinopsonization ofnanoparticlesand subsequent
phagocy-tosisbynon-parenchymalcellsoftheliverinvivo.Theshielding
effect of PEG prevents nanoparticles from reticuloendothelium
system(RES)uptake resultinginlong-circulatingcharacteristics
(Avgoustakis, 2004; Betancourtet al.,2009; Ioele,Cione, Risoli,
Genchi, &Ragno, 2005; Lu et al.,2009).The similar result has
beenreportedbyvanVlerkenetal.Theyfoundthatthepegylated nanoparticlesavoideduptakebyRES,therebyimproving circula-tiontimeofnanoparticles,andthenanoparticlesareretainedin thetumorforprolongedperiodoftime(vanVlerken,Duan,Little, Seiden,&Amiji,2008).Ontheotherhand,PEGhasbeenusedto improvesolubilityofchitosaninsimulatedgastricpHand physio-logicalpHviaalteringmolecularweightand/orsubstitutiondegree
ofPEG(Casettarietal.,2012;Jeong,Kim,Jang,&Nah,2008) Asialoglycoproteinreceptor(ASGPR) receivesmuch attention
in gene targeting and also plays as a model systemfor study-ing receptor-mediated endocytosis due to its high affinity and rapidinternalizationrate.ASGPRisanintegralmembraneprotein expressedonthesurfaceofparenchymalcellsofliverwithhigh density of1–5×105 receptors(Weigel &Yik,2002) Nanocarri-ers(e.g.,nanoparticles)withsurfacemodificationarenecessaryfor specifictargetingpurpose.Severalsugarligands(e.g.,galactose, N-acetylgalactosamine,mannose,lactose,fructose,etc.)haveproved
tointeractwithASGPRwithvariousextents.Galactosehasbeen provedrecognitionofASGPRthroughmanyinvitroandinvivo stud-ies.Wangetal.(2012)usedgalactoseandPEGmodifiedliposome
toencapsulatedoxorubicinwhichdemonstratedbettertargeting efficiencyandachieved94%tumorgrowthinhibition.Jiangetal preparedPEG-galactosefollowedbygraftedontotheaminogroup
ofchitosan-PEI IthadbettercellulartransfectionthanPEIafter intravenousinjection(Jiang etal.,2008).Chenetal.used lacto-bionicacidandglycyrrhetinicacidtopreparedual-ligandmodified chitosan.ItstransfectionefficiencyinASGPRhigh-expressed
BEL-7402cellswashigherthaninASGPR-freeLO2hepaticnormalcells (Chenetal.,2012)
http://dx.doi.org/10.1016/j.carbpol.2014.11.046
Trang 2duetosimpleandeasysyntheticprocedure.However,thepositive
chargeofC2-NH2groupplaysanimportantroleincomplexwith
negatively charged DNA for gene delivery Some studies were
designed to modifychitosan through C6-OHbut leave C2-NH2
groupavailableforDNAcomplexation.Jiang,Wu,Xu,Wang,and
Zeng(2011)usedC6-OHmodifiedchitosantocomplexwithvarious
weightratiosofDNA,andfoundthemolarratioofpolymer/DNA
20:1 expressed the highest cellular transfection The chemical
modificationofchitosanbygraftinglactobionicacid,asareceptor
ligand,throughC6-OHpositionofchitosanhasbeendemonstrated
(Lin,Chen,&Liu,2009;Lin,Chen,Liu,Chen,&Chang,2011)
Lacto-bionicacidisanendogenoussubstancepresentinthehumanbody
(Yu&vanScott,2004).Thechemicalstructureoflactobionicacid
containsagalactoseunitandagluconicacidunitlinkedbyether
linkage.Thecarboxylgroupofgluconicacidunitreactswiththe
aminogroupofchitosantoformanamidelinkage.Thelactobionic
acidgraftedchitosandemonstratedhighertransfectionefficiency
thanligand-freechitosan(45.3%vs19.8%)inASGPRoverexpressed
HepG2 cells Zhang et al (2009) grafted galactose onto C6-OH
followedbypegylationfromC2-NH2group.Theydemonstratedno cytotoxicityofmodifiedchitosaninHEK293kidneycancercells However,itwaslackofdatatoverifythefeasibleapplicationof thismodifiedchitosanindrugand/orgenedelivery
Thepresentstudywasaimedtodevelopahepatocyte-targeting non-viralpolymericnano-carrierforgenedelivery.Chitosanwas selectedasthemainpolymer.Inordertohavespecificliver tar-getingactivity,an ASGPRrecognized sugarmolecule, galactose, wasintroducedintoC6-OHofchitosan.Thehydrophilicmethoxy poly(ethylene glycol)(mPEG) orshort chain PEGdiacid (PEGd) wasgraftedontogalactosylatedchitosanfurtherthroughits
C2-NH2positiontoincreasesolubilityandstabilityofchitosaninvivo ThesynthesizedchitosanderivativeswerecharacterizedbyFTIR, NMRandGPC,andthegalactose,mPEGandPEGdgraftcontents weredetermined.ThegalactosylatedchitosangraftedwithmPEG
orPEGdwasappliedtocomplexwithplasmidDNA,andthe perfor-manceofpolymer/DNApolyplexwascharacterized.Theabilityof condensingnegativelychargedplasmidDNAbymodifiedchitosan, thestabilityofpolymer/DNApolyplexanditscellulartransfection wereevaluated
O
NH HO
O
OH
O
HO
O OH HO
OH HO
O
O
O
O
O
O
HO O
O
OH n
(A) chitosan-galactose
O
HO O
OH
OH
HO
chitosan
O OH HO
OH OH HO
O
HO O
OH
O
HO
O OH HO
OH HO
mPEG
O
O OH
m
O
NH HO
O
OH
O
HO
O OH HO
OH HO
O
O H m
Trang 32 Materials and methods
2.1 Materials
Lowmolecularweightchitosan(CS,Mw260kDa,Mn 72kDa,
deacetylationdegree76.3±2.1%)andpoly(ethyleneglycol)diacid
(PEGd,Mn 600Da) were from AldrichChemical Company, Inc.,
(WI,USA).Methoxypoly(ethyleneglycol)(mPEG,MW5,000Da),
borontrifluoridediethylether(BF3•OEt2),andanthronewerefrom
FlukaChemicalCompanyInc.(Buchs,Switzerland).d(+)-Galactose
(99+%)andN-hydroxysuccinimide(NHS)werefromAcros
Organ-ics Co Inc (Geel, Belgium) Sodium nitrite (NaNO2) was from
Showa Chemical Co Ltd (Tokyo, Japan) Sodium
cyanoborohy-dride(NaCNBH3,95%)wasfromAlfaAesaraJohnsonMattheyCo
Inc.(Massachusetts,USA).1-Ethyl-3-(3-dimethylaminopropyl
car-bodiimidehydrochloride(EDC)wasfromTCI ChemicalIndustry
Co.Ltd.(Tokyo,Japan).Minimumessentialmedia(MEM)wasfrom
BiologicalIndustriesIsraelBeit-HaemekLtd.(BeitHaEmek,Israel)
Plasmidencodingenhancedgreenfluorescentprotein(pEGFP-N1,
4.7kb) was kindly provided by Professor Jiin Long Chen from
NationalDefenseMedicalCenterinTaiwan.TheHepG2cancercell
linewasagiftfromDr.Hui-LinWuinHepatitisResearchCenterof
NationalTaiwanUniversityHospitalinTaiwan
2.2 Synthesisofchitosan-basedpolymers
Fig.1(A)showstheprocedurestosynthesizechitosan-galactose
(Chitosan(1))(Linetal.,2009,2011).Chitosanwasdeacetylated
inNaOHaqueoussolution(50%w/v)at140◦Cfor4hfollowedby
depolymerizedin0.1Msodiumnitriteaceticsolutionatroom
tem-peraturefor3h.Theobtaineddeacetylateddepolymerizedchitosan
(DADPCS)wasreactedwithgalactoseatfeedmolarratio1:2.5in
themixtureoftetrahydrofuran(THF)andborontrifluoridediethyl
etherate(BF3•OEt2)at60◦C underN2 for24h Thesolventwas
removedbyrotaryevaporation,andthemixturewasdialyzed(MW
cut-off500-1000Dalton)followedbyfreezedried
Fig.1(B)showstheprocedurestosynthesize
chitosan-galactose-mPEG(Chitosan(2)).mPEGwasdissolvedina mixtureofDMSO
and chloroform (10:1, v/v) followed by reacting with acetic
anhydride at room temperature for 9h The ether was added
to precipitate the product mPEG-CHO which was collected
after filtration The mPEG-CHO was dialyzed (MW cut-off
500-1,000Da)andfreezedried.Chitosan(1)waspreviouslydissolved
in a mixture of 2% acetic acid and methanol (1:1 v/v)
mPEG-CHOin deionized water was slowlyadded into and reactedat
room temperature for 3h followed by adding NaCNBH3
aque-oussolution underN2 for further18h Thefeedmolar ratioof
Chitosan(1):mPEG-CHO:NaCNBH3 was1.0:0.6:4.5.Thereaction
solutionwasconcentrated by rotary evaporation,and the
mix-turewasdialyzed(MWcut-off6000-8000Da)followedbyvacuum
dried
Fig.1(C)showstheprocedurestosynthesize
chitosan-galactose-PEGd (Chitosan(3)) Chitosan(1) waspreviously dissolvedin 1%
aceticacidsolution.PEGd,NHSandEDCwereslowlyaddedinto
andreactedatroomtemperaturefor24h Thefeedmolarratio
ofChitosan(1):PEGd:NHS:EDCwas1:7:7:7.Thereactionsolution wasdialyzed(MWcut-off6000-8000Da)followedbyfreezedried TheobtainedChitosan(3)waswashedbyacetoneforthreetimes followedbyvacuumdried
2.3 Characterizationofchitosan-basedpolymers TheobtainedChitosan(1), Chitosan(2), and Chitosan(3) were characterizedbyFTIRand1HNMR,themolecularweights were analyzedbyGPC.Thegalactosylationratiointermsofweight per-centage(Wg%)wascalculatedaccordingtoEq.(1).Thegalactose degreeofsubstitution(DSg%)ofChitosan(1),Chitosan(2),and Chi-tosan(3)weredeterminedbyanthrone–sulfuricacidcolorimetric assay(Laurentin&Edwards,2003)andcalculatedaccordingtoEqs (2),(3),and(4),respectively
GalactoseWg(%)= galactosesampleweightweightinthesample×100% (1)
galactoseDSg1(%)= (galactoseweight) /
Mw galactose
(sampleweight−galactoseweight) / (Mw DADPCS monomer)×100% (2)
Mw galactose
(sampleweight−galactoseweight−mPEGweight) / (Mw DADPCS monomer)×100% (3)
Mw galactose
(sampleweight−galactoseweight−PEGdweight) / (Mw DADPCS monomer)×100% (4)
ThemPEGdegreeofsubstitution(DSmPEG%)ofChitosan(2)was calculatedbyEq.(5)basedon1HNMRdata,andthepegylation weightpercentage(WmPEG%)wascalculatedbyEq.(6)
DSmPEG(%)= (areaofpeakc)3.5ppm/3
W mPEG (%) = DSmPEG × M w mPEG
(DS mPEG × M w mPEG ) + (100% × M w monomer ) +
DS g2 × M w galactose
Similarly,thePEGd degreeofsubstitution(DSPEGd%)and the pegylationweightpercentage(WPEGd%) ofChitosan(3) were cal-culatedbyEqs.(7)and(8),respectively
DSPEGd(%)= (areaofpeakc)4.15ppm/2
(areaofpeakd)3.1ppm
(DS PEGd ×M w PEGd ) + (100% × M w DADPCS monomer )+
DS g3 × M w galactose
2.4 Gelpermeationchromatography(GPC) Themolecularweightaswellasmolecularweightdistribution
interms ofpolydispersityofmodified chitosanwasdetermined
bygelpermeationchromatography(GPC)equippedwitha refrac-tiveindexdetector(ShimadzuRID-10A,Japan).Twolinearcolumns (UltrahydrogelTM 500and DP 120, 7.8×300mm, Waters) were appliedand acetatebufferedsolutionatpH5.0wasusedasthe elutingsolventataflowrateof0.8mL/minat35◦C.The calibra-tioncurvewasconstructedusingdifferentmolecularweightsof poly(ethyleneglycol)standards.Themolecularweightofmodified chitosanwasre-calculatedfromthecalibrationcurvebasedonthe measuredretentiontime
Trang 42.5 Galactosedetermination
Thecontentofgalactosegraftedontochitosanwasmeasured
bycolorimetric assayusing anthronesulfuric acid(Laurentin&
Edwards,2003).Severalknownconcentrationsofgalactose
solu-tionswereplacedina96-wellpre-cooledat4◦C.Thefreshprepared
anthrone–sulfuricacidinanicebathwasaddedintothe96-well
The96-wellwasheatedat90◦Cfor6minfollowedbycooledto
roomtemperature.Theabsorbancewasdeterminedby
spectropho-tometerat630nm.Thecalibrationcurvewasconstructedbased
onseveralconcentrationsofgalactoseandtheirabsorbance.The
polymersampleswerepreparedaccordingtothesameprocedure,
andthecorrespondingconcentrationwasre-calculatedfromthe
calibrationcurvebasedonthemeasuredabsorbance
2.6 Cytotoxicityofgalactosylatedandpegylatedchitosan
Thecytotoxicity ofChitosan(1), Chitosan(2), and Chitosan(3)
wasinvestigated.HepG2cellswereculturedinthemodifiedEagle’s
mediumcontaining10%fetalbovineserum,sodiumbicarbonate,
nonessential amino acids and sodiumpyruvate The cells were
seeded in a 96-well plate at a density of 9000 cells per well
andmaintainedinahumidifiedincubatorat37◦Cin5%CO2 for
24h.Serialdilutionsofpolymersolutioninculturedmediumwere
addedintoeachwellandincubatedat37◦Cfor24h.Thecultured
mediumwithoutpolymersolutionwasthecontrol.Themedium
wasremoved,andtheMTTsolution
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumwasaddedandincubatedat37◦Cfor4h
(Ciapetti,Cenni,Pratelli,&Pizzoferrato,1993).Theresulting
forma-zanwassolubilizedindimethylsulfoxide,andtheabsorbancewas
measuredusinganenzyme-linkedimmunosorbentassay(ELISA)
reader(PowerWaveXS,BioTek,Winooski,VT)at570nm(Liu&
Lin,2013)
2.7 Preparationofpolymer/pDNApolyplex
Chitosan(1),Chitosan(2),andChitosan(3)werecomplexedwith
negativelychargedpDNAatvariousweightratiosof2:1,10:1and
20:1.Eachpolymerwaspreviouslydissolvedin1%aceticsolution
TheplasmidDNAwasdissolvedinsteriledistilledwaterfollowed
byslowlyaddedintopolymersolutionandstirredfor3min.The
resultingpolyplex wasstood for 3hat roomtemperature The
polyplexsolutionwascentrifugedat 16,000×g for30min.The
supernatantwasremoved,andthedistilledwaterwasaddedto
re-dispersethepolyplex.Theparticlesizeandzetapotentialwere
measuredbyusingZetasizernanoanalyzer(Nano-ZS90,Malvern
InstrumentsLtd.,Worcestershire,UK)at25◦C Themorphology
ofpolymer/pDNApolyplexwasobservedbytransmissionelectron
microscope(PhilipsTecnaiF30,Philips,Netherlands).Thestability
ofpolyplexwasevaluatedtheirparticlesizechangeduringstorage
at4◦Cfor28days
2.8 Transfectionofpolymer/pDNApolyplex
ThetransfectionofChitosan(1)/pDNA,Chitosan(2)/pDNA,and
Chitosan(3)/pDNApolyplexwasevaluatedinASGPRoverexpressed
HepG2cancercells.TheHepG2cancercellswereseededina
6-wellplateatadensityof6×105cells/wellandincubatedat37◦C
for 24h After that the medium was removed, the serum-free
MEMmediumcontainingpolymer/pDNApolyplexornakedpDNA
wasaddedintoeachwellandincubatedfor24h.The
phosphate-bufferedsolution(PBS)wasaddedafterthemediumwasremoved
Thecellsuspensionwascentrifugedat 1200rpm for5min.The
cellswerecollectedandresuspendedinpH7.4PBSforflow
cyto-metricanalysisinthefluorescencechannelFL-1atanexcitation
wavelength488nmandanemissionwavelength530nm.Atotal
Table 1
Chitosan-galactose
Chitosan-galactose-mPEG
Chitosan-galactose-PEGd
M w (Da) 6200 8500 7600
M n (Da) 4000 5500 5200
W g (%) 16.7 13.2 13.8
DS g (%) 18.4 27.3 28.2
W PEG (%) – 42.6 52.8
DS PEG (%) – 3.7 43.5
of10,000cellswereanalyzedforeachsample,andtheupperlimit
ofbackgroundfluorescencewassetnomorethan1%.Datawere presentedasmean±standarddeviation.Comparisonbetweentwo groupswasanalyzedbyStudent’st-test,andthedifferencewas consideredsignificantatp<0.05or0.01
3 Results and discussion
3.1 Characterizationofchitosan-galactose(Chitosan(1)) Fig.2(C)showsthe1HNMRspectrumofChitosan(1).TheMW andgalactosylationdataofChitosan(1)arelistedinTable1.The
Mw,MnandpolydispersityofChitosan(1)were6200Da,4000Da, and1.56,respectively.Thecorrespondinggalactosegraftingweight percentage(Wg(%))anddegreeofsubstitution(DSg1(%))were16.7% and18.4%,respectively
3.2 Characterizationofchitosan-galactose-mPEG(Chitosan(2)) PEG plays an important role in preventing nanoparticles aggregationand avoiding nanoparticleseliminated byRES The galactosylatedchitosanwasfurtherpegylatedbymPEG,andthe relevantcharacterizationdataofChitosan(2)aresummarizedin Table 1 The corresponding Mw, Mn and polydispersity of Chi-tosan(2)were8500Da,5500Da,and1.54,respectively.Fig.2(A) shows the 1H NMR spectrum of Chitosan(2) The peaks a at 3.6–4.0ppmwereassignedtoC3–C6protonsofchitosanandthe protonsofgalactose,andpeakdat3.2ppmwasassignedtoC2–H
ofchitosan.Thepeakbat3.6–3.8ppmwasassignedtotheprotons
ofmPEGrepeatunits( CH2 CH2 O ),andpeakcat3.5ppmwas assignedto OCH3ofmPEG.TheWg(%)andDSg2(%)ofgalactose cal-culatedbyEqs.(1)and(3)were13.2%and27.3%,respectively.The mPEGgraftingweightpercentage(WmPEG%)calculatedbyEq.(5) was42.6%,andthecorrespondingdegreeofsubstitution,DSmPEG%, calculatedbyEq.(4)was3.7%basedontheintegrationareaofpeak
candpeakdin1HNMRspectrum.Thedegreesofsubstitutionof galactoseandmPEGofmPEGylated-galactosylated-chitosan devel-oped byZhang et al.(2009) were0.09% and 0.3%,respectively, which weremuch lowerthan ours It seemed that thecurrent methodappliedtograftgalactoseandmPEG ontochitosan was moreefficientintermsofhighergraftingvaluesthantheirs 3.3 Characterizationofchitosan-galactose-PEGd(Chitosan(3)) AnotherapproachwasdesignedtopegylateChitosan(1)with short chain PEG diacid(MW 600Da), and therelevant charac-terization data of Chitosan(3) are summarized in Table 1 The corresponding Mw, Mn and polydispersity of Chitosan(3) were
7600Da,5200Da,and1.46,respectively.Fig.2(B)showsthe1H NMRspectrumofChitosan(3).Thepeakcat4.15ppmandpeakb
at3.6ppmwereassignedtotheC Hnextto COOHofPEGdiacid andtherepeatunits( CH CH O )ofPEGdiacid.Thepeaks
Trang 5Fig 2.The 1 H NMR spectra of (A) chitosan-galactose-mPEG, (B) chitosan-galactose-PEGd, (C) chitosan-galactose, (D) mPEG, and (E) PEG diacid.
Trang 6Polymer concentration (µg/mL)
500 250 150 100 50 25 15 5 1
0
20
40
60
80
100
120
chitosan-galactose-mPEG chitosan-galactose-PEGd
aat3.7–3.9ppmwasassignedtoC3–C6protonsofchitosanand
theprotonsofgalactose,andpeakdat3.1ppmwasassignedto
C2–Hofchitosan.ThegalactosegraftingWg(%)andDSg3(%)were
13.8%and28.2%,respectively.Theweightpercentageofpegylation
(WPEGd(%))was52.8%,andthecorrespondingDSPEGd(%)was43.5%
basedontheintegrationareaofpeak c and peakdin1HNMR
spectrum
3.4 Cytotoxicityofgalactosylatedandpegylatedchitosan
Fig.3illustratesthecellularviabilityofChitosan(1),Chitosan(2),
andChitosan(3) inHepG2 cells.The cytotoxicityofgrafted
chi-tosan wassimilarirrespective ofthe presence of PEGand PEG
chain length,and there wereat least 80% cells viable at
poly-mer concentration ≤5g/mL All of the grafted chitosan had
IC50correspondingto50%cytotoxicityhigherthan500g/mL.It
indicatedthatthegalactosylated-pegylated-chitosanhadlow
cyto-toxicityandwasmuchsafebeingusedinvivo.Kim,Shin,andLee
(1999)reportedthatthecytotoxicityofPEGwithmolecularweight
greaterthan3000Dawasignorable.Similarly,Maoetal.(2005)
foundthatthelowcytotoxicityofPEG-conjugated-chitosanwas
observedin PEG Mw 5000Da rather than550Da Nevertheless,
therewasnodifferenceincytotoxicitybetweenmPEG(5000Da)
andshortchainPEGdiacid(600Da)graftedchitosaninourcurrent
study
3.5 Characterizationofpolymer/DNApolyplex
Thegalactosylated-pegylated-chitosanwasappliedasa DNA
delivery carrier Complex of cationic chitosan and negatively
charged plasmid DNA spontaneously formed polyplex due to
electrostatic interaction.Fig.4(A) illustrates theparticlesize of
Chitosan(1)/pDNA,Chitosan(2)/pDNA,andChitosan(3)/pDNAwith
various polymer/DNA weight ratios The particle size of
Chi-tosan(2)/pDNA polyplex with polymer/pDNA weight ratio 2:1,
10:1,and 20:1 was 159.9±43.0, 104.6±8.1, and 98.7±6.6nm,
respectively.ThecompactionofDNAbyChitosan(2)was
promi-nent when polymer/DNA weight ratio wasincreased from 2:1
to10:1wheretheparticlesizewassignificantlydecreased
Fur-ther increase in polymer/DNA weight ratio to 20:1 did not
change particle size too much The sterically repulsive nature
of mPEG protected Chitosan(2)/pDNA from secondary
aggrega-tionandformedpolyplexwithreliableparticlesizeintherange
of100–200nm.Thesimilar phenomenonhasbeen reportedby
chitosan-gala
ctose/DNA chitosan-gala
ctose-mPEG /DNA
chitosan-gala
ctose-PEGd/DNA
0 100 200 300 400 500 600 700
800
2:1 10:1 20:1
chitosan-gala
ctose/DNA chitosan-gala
ctose-mPEG /DNA
chitosan-gala
ctose-PEGd/DNA
0 10 20 30 40 50 60
70 2:1 10:1 20:1
(B) (A)
Kataoka,Harada,and Nagasak(2001)wherethepolyionic PEG-poly(l-lysine) block copolymer was complexed with positively chargedpDNA.TheymentionedthatthePEGcoronasurrounded
onmicellesurfacedecreasedthelocaldielectric constantwhich facilitated DNA compacted by PEG–PLys However, only the 2:1(w/w) polyplex of Chitosan(3)/pDNA and Chitosan(1)/pDNA had particle size less than 200nm The increase of polymer (e.g.,polymer/DNA10:1 and20:1)was failtosufficiently com-pact DNA into polyplex of Chitosan(3) and Chitosan(1) which resulted in quite large in particle size The lack of steric pro-tectionbythesetwopolymersaccountedfor resultingpolyplex with quite large size All of these results implied that the compaction of DNA by grafted chitosan was in order of Chi-tosan(2)/pDNA>Chitosan(3)/pDNA>Chitosan(1)/pDNA, and the bestDNAcompactionwasachievedbyChitosan(2).The morphol-ogyofChitosan(2)/pDNApolyplexisillustratedinFig.5
Fig.4(B)illustratesthezetapotentialofpolyplexwithvarious polymer/DNAweightratios.Allpolyplexpossessedpositivecharge characterinorderofChitosan(2)/pDNA(+20–30mV)<Chitosan(3)/ pDNA(+40–50mV)<Chitosan(1)/pDNA (+45–60mV) The mPEG polymerchainssurroundedonthepolyplexsurfacediminishedthe positivechargeofchitosanresultinginthelowestzetapotential
ofChitosan(2)/pDNApolyplex.ThechainlengthofmPEGpolymer waslongerthan PEGdiacidwhere mPEGformedbettersurface
Trang 7Fig 5.The TEM image of chitosan-galactose-mPEG/pDNA polyplex with
coverageonChitosan(2)/pDNApolyplex.Ontheotherhand,the
shorterchainlengthofPEGdiacidexertedlesssurfacecoverage
thanmPEGandresultedinthezetapotentialofChitosan(3)/pDNA
higherthanChitosan(2)/pDNAbutlessthanChitosan(1)/pDNA
3.6 Stabilityofpolyplex
Fig.6illustratesthestability intermsofpercentageof
parti-clesizechangeofpolyplexafterstorageat4◦Cfor28days.Most
ofChitosan(2)/pDNAandChitosan(3)/pDNApolyplexmaintained
theirparticlesizeattheend of 28days except20:1(w/w)
Chi-tosan(3)/pDNApolyplex.Itloststabilityafterstoragefor21days
wherethepolyplexwasaggregatedinterms ofenlarging
parti-clesizemuch.AlloftheseresultsindicatedthatChitosan(2)was
not only capable of condensing plasmid DNA but also formed
stablepolyplexascomparedtoChitosan(1)andChitosan(3).The
presenceofmPEGofChitosan(2)playedanimportantrolein
pre-venting polyplexaggregation and maintainingits stablenature
wherethehydrophilicPEGchainssurroundedontheoutershell
ofthepolyplexandextendedintheaqueousenvironmenttoexert
shieldingeffect(Betancourtetal.,2009;Linetal.,2009;Luetal.,
2009)
3.7 Transfectionofpolyplex
Fig 7 illustrates the transfection efficiency of polyplex in
asialoglycoproteinreceptor (ASGPR)overexpressedHepG2 cells
The transfection of naked plasmid DNA (pEGFP-N1) was
simi-lar to the negative control (MEM medium only) However, all
of the polyplex enhanced pDNA cellular transfection as
com-paredtonakedDNAinorderofChitosan(1)/pDNA>Chitosan(3)/
pDNA>Chitosan(2)/pDNA.Increaseinpolymer/DNAweightratios
of Chitosan(1)/pDNA polyplex from 2:1 to 20:1 prominently
increased transfection efficiency in terms of producing more
greenfluorescentproteinsinASGPRoverexpressedHepG2cells
This provided the evidence to ensure the specific targeting of
galactose to ASGPreceptor The galactose grafting weight
per-centage(Wg%)ofChitosan(1),Chitosan(2) andChitosan(3)were
16.7, 13.2 and 13.8%, respectively Although the grafted
galac-tose of Chitosan(1) was similar to the other two kinds of
galactosylated-pegylated-chitosan,itsgalactosemoietywasfully
exposed and specifically bound to ASGP receptor to enhance
cellulartransfectionthemost.Nevertheless,theshieldingeffect
0 20 40 60 80 100 120 140 160
2:1 10:1 20:1
chi tosan-galac tose/pDNAweight rao
7 da y 14 day 21 da y 28 day
0.27 0.27 0.26 0. 0.22 0.22 0.27 0.38 0. 0.27 0.27
0 20 40 60 80 100 120 140 160
2:1 10:1 20:1
chitosan-galactose-mPE G/pDNA weight ra o
7 day 14 da y 21 day 28 day
0.22 0. 022 0 0. 0 0 0. 0.28 0.28 0.25 0.
0 20 40 60 80 100 120 140 160
chi tosan-galactose -PEG d/pDNA weight rao
7day 14day 21day 28 day
0.35 0 0.32 0 0.34 0 0 0.26 0.26 0.29 0.32 0.32
(A)
(B)
(C)
of mPEG on the surface of Chitosan(2)/pDNA polyplex dimin-ishedthespecifictargetingabilityofgalactosetoASGPreceptor resulting in the lowest cellular transfection in HepG2 cells as compared to the other polyplex On the other hand, the Chi-tosan(3)/pDNApolyplexwascoveredbyshortchainPEGdiacid TheshieldingeffectofPEGdiacidwasnotsoprominentasmPEG whichaccountedforthecellulartransfectionofChitosan(3)/pDNA polyplex higher than Chitosan(2)/pDNA but lower than Chi-tosan(1)/pDNA
Trang 8ol
Nake
d DNA ch
san-g alacto se
ch
san-g alacto
se-m
PEG
ch
san-g alacto se
EGd
0
5
10
15
20
25
30
35
40
2:1
10:1
20:1
**
**
**
*
**
**
**
**
**
4 Conclusion
The galactosylated-pegylated-chitosan with
asialoglycopro-teinreceptor targeting abilitywasdeveloped for genedelivery
The chitosan was chemically grafted by galactose and
differ-entchain lengthsof hydrophilic methoxypoly(ethylene glycol)
or poly(ethylene glycol) diacid The concentration of grafted
chitosan corresponding to 50% cytotoxicity was higher than
500g/mL The positively charged grafted chitosan formed
polyplex with negatively charged plasmid DNA, and the
com-paction of DNA by grafted chitosan was in order of
Chi-tosan(2)/pDNA>Chitosan(3)/pDNA>Chitosan(1)/pDNA All
poly-plexenhanced DNAcellulartransfection ascompared tonaked
DNA.AlthoughChitosan(2)/pDNApolyplexmaintaineditsparticle
sizeforlongesttime,theshieldingeffectofmethoxypoly(ethylene
glycol)diminished the specific targeting ability of galactose to
asialoglycoproteinreceptorresultinginthelowestcellular
trans-fectioninHepG2cells.Throughthisstudyelucidatedtheroleof
poly(ethyleneglycol)inchitosan-basedpolyplexstabilityand
cel-lulartransfection
Acknowledgments
ThisworkwassupportedbyNationalScienceCouncilTaiwan
(NSC102-2320-B-002-007-MY3).TheauthorsthankDr.FuHsiung
ChangfortheZetasizer,Dr.JiinLongChenforplasmidDNA,andDr
HuiLinWuforHepG2cellline
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