Abstract
Dimeric cationic surfactants (gemini-type) are a group of amphiphilic compounds with potential use in gene therapy as effective carriers for nucleic acid transfection (i.e., siRNA, DNA, and plasmid DNA). Our studies have shown the formation of lipoplexes composed of alkanediyl-α,ω-bis[(oxymethyl)dimethyldodecylammonium] chlorides and selected 21-base-pair nucleic acid (dsDNA and siRNA) oligomers. To examine the structure and physicochemical properties of these systems, optical microscopy, circular dichroism spectroscopy (CD), small-angle X-ray scattering of synchrotron radiation (SR-SAXS), and agarose gel electrophoresis (AGE) were used. The lengths of spacer groups of the studied surfactants had a significant influence on the surfactants’ complexing properties. The lowest charge ratio (p/n) at which stable lipoplexes were observed was 1.5 and the most frequently occurring microstructure of these lipoplexes were cubic and micellar phases for dsDNA and siRNA, respectively. The cytotoxicity tests on HeLa cells indicated the non-toxic concentration of surfactants to be at approximately 10 µM. The dicationic gemini surfactants studied form complexes with siRNA and dsDNA oligomers; however, the complexation process is more effective towards siRNA. Therefore these systems could be applied as transfection systems for therapeutic nucleic acids.
Highlights
Gene therapy offers many methods for insertion of DNA, RNA fragments, or chimeric DNA–RNA systems into target cells [1,2]
The mixtures of siRNA and dsDNA oligomers with C12NCn gemini surfactants were first analyTzheedmuisxintugreospotifcsailRmNiAcraonsdcodpsyD(NOAMo).liIgnotmheerhsiwghitehstCc1o2nNcCenntgraetmioinnsi soufrtfhaectaamntpshwiperheilfier(spt /anna=ly2–z4ed), tuhseintgesotpedticmalixmtuicrreosswcoepryer.aInnstphaerehnigt hdeuset ctoontcheenftorarmtioantisoonfothf espaamtipahl isptrhuilcetu(pre/ns v=is2i–b4l)e, ethveenteostneda mmiaxctruorsecsowpiecrescnaolet t(rsaenesFpiagruenret d2)u.eTthoistheefffeocrtmisataiornesoufltspoafttihalessterulfc-oturrgeasnvizisaitbiolenepvreoncoesnsaomf tahcerossicRoNpAic oscradlesD(sNeeAFpigoulyrean2i)o. nTsh, iasnedfftehcet ipsoasirteivsuellyt ocfhtahrgeesdelsf-uorrfgaactnainztastiwonithpirnoctehses loipf othpelesxieRsNfoArmoreddsiDn NthAe psoolluytainoino.ns, and the positively charged surfactants within the lipoplexes formed in the solution
Our gel electrophoresis experiments and circular dichroism studies showed that for the formation of stable C12NCn lipoplexes, a specific p/n ratio is required for both siRNA and dsDNA
Summary
Gene therapy offers many methods for insertion of DNA, RNA fragments, or chimeric DNA–RNA systems into target cells [1,2]. Dicationic surfactant molecules in appropriate conditions (aqueous solution, pH, and ionic strength), and in the presence of nucleic acids, can form lipoplexes organized in supramolecular microstructures like hexagonal, lamellar, or cubic phases [13,14,15,16,17]. 33 ooff 1187 where n+ and n− are the numbers of positive and negative charges, csurf and coligo are the wcohnecrennt+raatnidonns−oafreththeesnuurmfabcetrasnotfapnodsitsiviReNanAd noergdatsiDveNcAha,rrgeesp, cescutrfivaenldyc. The mixtures of siRNA and dsDNA oligomers with C12NCn gemini surfactants were first analyTzheedmuisxintugreospotifcsailRmNiAcraonsdcodpsyD(NOAMo).liIgnotmheerhsiwghitehstCc1o2nNcCenntgraetmioinnsi soufrtfhaectaamntpshwiperheilfier(spt /anna=ly2–z4ed), tuhseintgesotpedticmalixmtuicrreosswcoeprye (nOoMt t)r.aInnstphaerehnigt hdeuset ctoontcheenftorarmtioantisoonfothf espaamtipahl isptrhuilcetu(pre/ns v=is2i–b4l)e, ethveenteostneda mmiaxctruorsecsowpiecrescnaolet t(rsaenesFpiagruenret d2)u.eTthoistheefffeocrtmisataiornesoufltspoafttihalessterulfc-oturrgeasnvizisaitbiolenepvreoncoesnsaomf tahcerossicRoNpAic oscradlesD(sNeeAFpigoulyrean2i)o. NTsh, iasnedfftehcet ipsoasirteivsuellyt ocfhtahrgeesdelsf-uorrfgaactnainztastiwonithpirnoctehses loipf othpelesxieRsNfoArmoreddsiDn NthAe psoolluytainoino.ns, and the positively charged surfactants within the lipoplexes formed in the solution. BBiinnddiinngg CCaappaacciittyy ooff CC1122NNCCnn wwiitthh ssiiRRNNAA aanndd ddssDDNNAA TThhee mmixixtutureressofosfursfuarcftaacnttasnwtsithwsiethlecsteeldecstieRdNAsiRoNr dAsDoNr AdsoDligNoAmeorlsi,gpormepearrse, dparsepabaorevde daesscarbiboevde, wdeesrcerisbeepda,rwateerde isnepthaeraategdarionsethgeeal,gaanrodsethgeefil,naanldefftheectfsinoafleeleffcetcrtospohfoereletcictrsoepphaorraettiiocnsaeprearsahtoiownnairne sFhigouwrne i3na.FiNguurcele3iac.aNciudcloeliicgaocmiderosliignotmheerns aintivtheefonramtivaerfeoprmolyaarenipoonlsy;atnhieornesf;otrhee, rtehfeoyres,htohueyldshmoouvlde mtowovaerdtsowthaerpdosstihtieveppoosilteivine tphoeleeleinctrtohpeheolreecttircogpehlo[3re3t,i3c4]g.eTl h[3e3c,o3m4]p. BBiinnddiinngg CCaappaacciittyy ooff CC1122NNCCnn wwiitthh ssiiRRNNAA aanndd ddssDDNNAA TThhee mmixixtutureressofosfursfuarcftaacnttasnwtsithwsiethlecsteeldecstieRdNAsiRoNr dAsDoNr AdsoDligNoAmeorlsi,gpormepearrse, dparsepabaorevde daesscarbiboevde, wdeesrcerisbeepda,rwateerde isnepthaeraategdarionsethgeeal,gaanrodsethgeefil,naanldefftheectfsinoafleeleffcetcrtospohfoereletcictrsoepphaorraettiiocnsaeprearsahtoiownnairne sFhigouwrne i3na.FiNguurcele3iac.aNciudcloeliicgaocmiderosliignotmheerns aintivtheefonramtivaerfeoprmolyaarenipoonlsy;atnhieornesf;otrhee, rtehfeoyres,htohueyldshmoouvlde mtowovaerdtsowthaerpdosstihtieveppoosilteivine tphoeleeleinctrtohpeheolreecttircogpehlo[3re3t,i3c4]g.eTl h[3e3c,o3m4]p. lTehxeaticoonmepffilecxieanticoineseofffisciiReNncAieasnodf sdisRDNNAAaonldigdosmDeNrsAbyoltihgoemteesrtsedbyC1th2eNtCesntesdurCfa1c2tNanCtsn, ssuturdfaicetdanintst,hsetugdeileedleicntrtohpehgoerleesliescetxroppehriomreesnits, wexepreerciommenpta,rwederine ocordmepr atoreiddeinntiofrydaersytsoteidmenwtiitfhy tahseylsotwemestwpi/tnhvthaleuleosw(Feisgtupr/en3vba,cl)u.eIst w(Faigsushreow3bn,ct)h. aItt wthaeslesnhgotwh noftthhaetstuhrefalcetnagntthspoafctehregsrouurfpacstiagnntifiscpaanctelyr ignrflouuepncsiegsnthifeicnaunctllyeicinafcliudesnbciensdtihneg npurocpleeirctiaecsibdys baminpdhinipghpilricopmeortleiecsulbeys. aFmorpthhiepghriloiuc pmooflaemcuplehsi.phFoilrictmheolgercouuleps wofitahmshpohritpshpilaiccemrso(Cle1c2uNleCs 2w-Cit1h2NshCo7r)t, sthpearceerws a(sCn12oNsCig2n-iCfi1c2aNntCd7i)ff, etrheenrceewinasbinnodisniggnoifficsaiRntNdAifafenrdendcseDiNn Abinodliignogmoefrss,iRwNhAichancodmdpslDexNeAd oulsiugaolmlyerins, twhehipch/ncroamnpgelex1e.5d–2u.suFaolrlythine tshuerfpa/cntarnatnsgwe i1th.5–lo2n. gFoerr sthpeacseurrfgarcotuanptss (wi.eit.,htlhoenogenresspfraocmer gCr1o2uNpCs8(ti.oe.C, 1t2hNe Co1n2e)s, wfreomobsCer1v2eNdCa8smtoalCl d12eNcreCa1s2e),inwtheeobbinsedrivnegdeffia csimenaclyl odfedcrseDaNseAinreltahteivbeitnodtihnagt eofffsiciRieNncAy, wofhdicshDwNaAs vreislaibtilveeattop/tnha=t 3o.fCsoiRmNpAar,iswohnicohf twheasgevliseilbecletraotphpo/nre=sis3.reCsoumltspaalrsiososnhoowf tehdethgaetl eCl1e2ctNroCpnhsourerfsaisctraensutsltws iatlhsoansheovwenednuthmatbCer1(2nN) Cofnmsuertfhayclteannetssewgimtheanntseivnetnhneusmpabceerr (gnr)ooufpmeexthhiyblietende sbeegttmerecnotsmipnltehxeinsgpapcreorpgerrotiuesp. exhibited better complexing properties
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