Novel cationic pentablock copolymers as non-viral vectors for gene therapy
- 格式：pdf
- 大小：471.99 KB
- 文档页数：14
Novelcationicpentablockcopolymersasnon-viralvectorsforgenetherapy
AnkitAgarwala,RobertUnferb,SuryaK.Mallapragadaa,*
aDepartmentofChemicalEngineering,IowaStateUniversity,3035SweeneyHall,Ames,IA-50011-2230,UnitedStatesbIowaCancerResearchFoundation,Urbandale,IA-50322,UnitedStatesReceived30August2004;accepted17November2004Availableonline19December2004
AbstractNewcationicpentablockcopolymersofpoly(diethylaminoethylmethacrylate)(PDEAEM),poly(ethyleneoxide)(PEO)andpoly(propyleneoxide)(PPO)—PDEAEM-b-PEO-b-PPO-b-PEO-b-PDEAEM—synthesizedinourlaboratorywereinvestigatedfortheirpotentialasnon-viralvectorsforgenetherapy.AgarosegelstudiesshowedthatthecopolymerseffectivelycondensedplasmidDNAtoformpolyplexes,andalsoprotectedplasmidsagainstnucleasedegradation.Lightscatteringandtransmissionelectronmicroscopywereusedtoanalyzetheapparentsize,molecularweightandmorphologyofthesepolyplexes.Lactatedehydrogenaseassaywasemployedtofindthecytotoxicitylimitsofthepolymersandpolyplexesonahumanovariancancercellline.ThepolymersshowedmuchlesscytotoxicitythancommerciallyavailableExGen500(linearpolyethyleneimine).Bychangingtherelativelengthsoftheblocksinthecopolymers,itwasfoundthatthecytotoxicityofthesecopolymerscouldbetailored.ThemicellarstructuresofthesecopolymersinaqueoussolutionsandtheirpH-sensitiveprotonationwereaddedadvantages.Invitrotransfectionefficienciesofthepolymersusinggreenfluorescentprotein(pEGFP-N1)andluciferase(pRL-CMV)reportergeneswerefoundcomparabletoExGen500.Besides,aqueoussolutionsofthesepentablockcopolymershavebeenshowntoexhibitthermodynamicphasetransitionsandthermoreversiblegelation,aqualitythatcouldallowsubcutaneous/intramuscularinjectionsofthesepolymersforcontrolledgenedeliveryovertime.D2004ElsevierB.V.Allrightsreserved.Keywords:Blockcopolymers;Injectable;Cationic;Non-viralvectors;Genetherapy;pHsensitive
1.Introduction
Non-viralgenetherapyusingcationiccopolymershasrecentlygainedincreasedinterestasapotentialtreatmentforcancerandseveralothergeneticdiseases[1–3].Itcanovercomeproblemsencoun-teredwithviral-basedtherapies,suchasimmunoge-nicity,toxicity,mutagenicityandpotentialdangerofoncogenicity[2].Non-viralgenetherapyinvolvingpolymersprovidesflexibilitytodesignacarrierhavingwelldefinedstructuralandchemicalproper-tiesonalargescale.Thepositivelychargedgroups0168-3659/$-seefrontmatterD2004ElsevierB.V.Allrightsreserved.doi:10.1016/j.jconrel.2004.11.022*Correspondingauthor.Tel.:+15152947407;fax:+15152942689.E-mailaddress:suryakm@iastate.edu(S.K.Mallapragada).JournalofControlledRelease103(2005)245–258www.elsevier.com/locate/jconrelGENEDELIVERYofthepolycationenableformationofbpolyplexesQwiththenegativelychargedphosphatesofDNAviaelectrostaticinteractions.ThisresultsinDNAcon-densation,protectionfromthenucleasedigestionandmoreefficientdeliveryofplasmidintothecell[4].Avarietyofpolycationshavebeenproposedandinvestigatedforpolyplexformation[5],suchaspoly-l-lysine(PLL)[6],polyethyleneimine(PEI)[7],polyamidoaminedendrimer[8],and(poly(2-dimethylamino)ethyl-methacrylate)(PDMAEMA)[9].However,thesesystemsareverytoxicandtheytendtoaggregateinvivo[10,11].Also,theyneedtobeadministeredrepeatedlyforsustainedgeneexpression.Aminemethacrylate-basedpolymershaveprevi-ouslybeenreportedasefficientcationiccondensingagentsforgenedelivery[12,13].WehavedesignednovelpentablockcopolymersofPDEAEMandPluronicsR,whicharetriblockcopolymersPEO-b-PPO-b-PEO[14,15].TheyretainthethermoreversiblegelationpropertiesofthetriblockPluronicsR[16,17],whileprovidingpH-sensitivegroupsforDNAcon-densationandendosomolysis[18].ThetertiaryaminegroupsofPDEAEMareresponsibleforDNAcondensationandprovidingpHbufferingcapacitytothepolymer.Also,asithasbeennotedbyFerrutietal.[19]andothers[20]thatmacromoleculeswithtertiaryaminegroupsexhibitlowertoxicitythanthosewithprimaryandsecondaryresidues,ourpentablockcopolymersareexpectedtobemorebiocompatiblethancommonlyusedvectorssuchasExGen500.Thesepentablockcopolymersformmicellesinaque-oussolutions[14]whichisanaddedadvantageasthemicellarstructureofPluronicsRhasbeenshowntofacilitatecellularentryandhasbeenfoundtosensitizemultidrugresistanttumors[21,22].Thecopolymerscanbemixedwiththetherapeuticgeneinanaqueousphaseatlowtemperatures(below48C)wheretheyexistassolsandcanforminjectablepolyplexes.Onsubcutaneous/intramuscularinjectionandsubsequentheatingtobodytemperatures,thecopolymersself-assembleintogels[14,15]thatcanactasreservoirsforsustained-releaseofpolyplexes.Theseinjectabledeliverysystemshaveseveraladvantagesoverothercommongenedeliverysystems,suchassimplepreparationwithoutorganicsolvents;thelackofsurgicalprocedurestoimplantmatrices;easystorageat48C;abilitytovarypolymerfractionstotailorandminimizecytotoxicity;andlastly,controlledreleaseofthepolyplexestocircumventrepeatedadministrationsneededwithotherpolymers.
