Synthesis and Characterization of Alkylated Poly(1-vinylimidazole) toControl the Stability of its DNA Polyion Complexes for Gene Delivery

Shoichiro Asayama,* Tomoe Hakamatani, and Hiroyoshi Kawakami

Department of Applied Chemistry, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo 192-0397, Japan.Received September 17, 2009; Revised Manuscript Received March 25, 2010

Poly(1-vinylimidazole) (PVIm) with alkylated imidazole groups has been synthesized as a pH-sensitive polycationto control the stability of its DNA polyion complexes for gene delivery. The resulting alkylated PVIm (PVIm-R)was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid-basetitration and solution turbidity measurements. Agarose gel retardation assay proved that the alkylated imidazolegroups worked as anchor groups to retain DNA. Pyrene fluorescence measurement showed that the hydrophobicdomain of the DNA complex with butylated PVIm (PVIm-Bu) increased after the protonation of imidazole groupsof the PVIm-Bu to enhance the membrane disruptive activity. The PVIm-Bu exhibited no significant cytotoxicityin spite of the existence of cationic groups. The resulting PVIm-Bu/DNA complexes easily released DNA, ascompared with the octylated PVIm, which was examined by competitive exchange with dextran sulfate. As aresult, the PVIm-R/DNA complexes mediated efficient gene delivery, and the gene expression depended on thelength and density of the alkyl chains. These results suggest that pH-sensitive PVIm-R’s control of the stabilityof DNA polyion complexes enhanced noncytotoxic gene delivery by the optimized alkylated imidazole groups.

INTRODUCTION

In gene delivery systems, the formation of polycation/DNApolyion complexes is a key factor for the new design of efficientdelivery (1-3). The polyion complexes on the cell plasmamembrane are internalized into acidic endosomal vesicles wherethey are subjected to a significant pH change from pH 7 to 5(4). Endosomal escape is one of the critical factors for efficientgene delivery. pH-sensitive polymers such as poly(ethylenimine)(PEI1), which is able to capture protons entering an endosome,have been used to achieve efficient release of the deliveredmaterial from endosomes (5-7). PEI induces swelling of theendosomes that leads to membrane disruption, that is, the protonsponge effect.

Recently, polymers modified with histidine or other moietiescontaining an imidazole group have shown significant enhance-ment of gene expression without increasing cytotoxicity com-pared with that of nonmodified polymers (8-12). In this case,histidine or other moieties containing an imidazole group havemade polycation/DNA complexes escape from an endosome bya proton sponge mechanism. The imidazole heterocyclesdisplaying a pKa around 6 possess buffering capacity inendosomal pH, inducing membrane destabilization after theirprotonation. The resulting imidazole groups facilitate the releaseof polycation/DNA complexes to cytosol.

However, we have already reported a poly(1-vinylimidazole)(PVIm) with several aminoethyl groups, that is, aminated PVIm(PVIm-NH2), for a pH-sensitive polycation to enhance cell-specific gene delivery (13). By using PVIm-NH2 as a pH-

sensitive DNA carrier, as well as a lactosylated poly(L-lysine)as a cell-targeting DNA carrier, the resulting ternary complexesspecifically mediate gene expression. Gene expression dependson our new concept that DNA ternary complexes dissociateligand polycations in response to endosomal pH (13, 14).However, PVIm-NH2/DNA binary complexes mediate nosignificant gene expression.

In this study, to develop PVIm-NH2 for the realization ofefficient gene expression, we have synthesized PVIm withseveral alkylated imidazole groups, that is, alkylated PVIm(PVIm-R). PVIm is a water-soluble homopolymer possessingmany imidazole groups. In spite of a large capacity for H+

buffering at endosomal pH, PVIm has difficulty in formingcomplexes with DNA at physiological pH because its imidazolegroups are negligibly charged at physiological pH. The intro-duced alkylated imidazole groups with a quaternary nitrogenatom are expected to work as new anchor groups to retain DNAand to control the stability of polyion complexes. The controlof the stability of the polyion complexes by the length anddensity of the alkylated imidazole groups has no precedent, tothe best of our knowledge. Consequently, pH-sensitive PVIm-R’s control of the stability of DNA polyion complexes isexpected to mediate efficient gene expression by optimizing thelength and density of the alkylated imidazole groups.

EXPERIMENTAL PROCEDURES

Materials. 1-Vinylimidazole (VIm), 1-bromobutane, 1-bro-mooctane, and pyrene were purchased from Aldrich ChemicalCo. (Milwaukee, WI). VIm was distilled under reduced pressure.2,2′-Azobis(2,4-dimethylvaleronitrile) (V-65) and bromoethanewere purchased from Wako Pure Chemical Industries, Ltd.(Osaka, Japan), and V-65 was recrystallized from ethanol.Iodomethane was purchased from Kanto Chemical Co., Inc.(Tokyo, Japan). PEI solution (Mn ) ∼60,000) and salmon testisdeoxyribonucleic acid (DNA) sodium salt were from SigmaChemical Co. (St. Louis, MO). All other chemicals of a specialgrade were used without further purification.

* To whom correspondence should be addressed. Tel: +81-42-677-1111 (ext.) 4976. Fax: +81-42-677-2821. E-mail: asayama-shoichiro@c.metro-u.ac.jp.

1Abbreviations: PEI, poly(ethylenimine); PVIm, poly(1-vinylimi-dazole); PVIm-NH2, aminated poly(1-vinylimidazole); PVIm-R, alky-lated poly(1-vinylimidazole); VIm, 1-vinylimidazole; V-65, 2,2′-azobis(2,4-dimethylvaleronitrile); GFC, gel filtration chromatography;FBS, fetal bovine serum; EtBr, ethidium bromide; RLU, relative lightunit; DS, dextran sulfate.

Bioconjugate Chem. 2010, 21, 646–652646

10.1021/bc900411m 2010 American Chemical SocietyPublished on Web 04/05/2010

Synthesis of PVIm-R. The synthetic route of PVIm-R isshown in Scheme 1. VIm (1) (300 µL) and V-65 (15.8 mg) asan initiator were dissolved in 2.7 mL of N,N-dimethylformamide(DMF). The radical polymerization reaction was carried out at45 °C for 1 day. After the reaction, the content was pouredinto a large excess of acetone, and the precipitate was dried inVacuo. The resulting polymer (2) (25 mg) and various amountsof alkyl halide (iodomethane, bromoethane, 1-bromobutane, or1-bromooctane) (3-100 µL) were dissolved in 2 mL of DMF.The reaction was carried out at 40 °C for 1-6 days accordingto each case. The reaction mixture was poured into a large excessof diethyl ether. The precipitate was dried in Vacuo and dissolvedin water. After dialysis against distilled water using a Spectra/Por 7 membrane (molecular weight cutoff ) 103), the resultingpolymer (3) was obtained by freeze-drying.

Gel Filtration Chromatography (GFC). GFC was carriedout using a JASCO PU-980 pumping system (Tokyo, Japan) atthe flow rate of 1.0 mL/min with a Shodex OHpak SB-804 HQcolumn (Showa Denko K. K., Tokyo, Japan). The aqueoussolution containing 0.5 M CH3COOH and 0.2 M NaNO3 wasused as a mobile phase. One hundred microliters of 1 mg/mLsamples were injected into the column. Eluate was detected bya refractive index detector (RI-1530, JASCO). Calibration wasmade with polyethylene glycol standards.

1H NMR Spectroscopy. Each polymer (3 mg) was dissolvedin 700 µL of D2O (99.8 atom % deuterium; Acros, NJ). The 1HNMR spectra (400 MHz) were obtained by a JEOL JNM-AL400spectrometer (Tokyo, Japan).

Acid-Base Titration and Turbidity Measurement ofPVIm-R. To 1.5 mL of an aqueous solution of the polymer(3.3 mg/mL) was added a 1 M HCl solution, and the acidicpolymer solution (pH 4) was titrated with a 0.2 M NaOHsolution. The pH value was checked with a pH meter (modelF-52T, Horiba, Kyoto, Japan). The titration was carried out bythe stepwise addition of 0.2 M NaOH and stopped at pH 10.The turbidity of the solution during the titration was measuredby monitoring the absorbance at 500 nm with a spectropho-tometer (model Ubest-55, JASCO, Tokyo, Japan).

Agarose Gel Retardation Assay. Salmon testis DNA wasdissolved in PBS (-) at 1.1 mg/mL. The resulting DNA stocksolution was added to the polymer solutions in 50 mM sodiumphosphate buffer (pH 7.5 or pH 6.0) at various polymer/DNAratios. The final diluted concentration of DNA was adjusted to66.7 µg/mL. After 30 min of incubation at room temperature,each sample (corresponding to 1 µg of DNA) was mixed witha loading buffer and loaded onto a 1% agarose gel containing1 µg/mL of ethidium bromide (EtBr). Gel electrophoresis wasrun at room temperature in 50 mM sodium phosphate buffer

(pH 7.5 or pH 6.0) at 50 V for 15 min. The DNA bands werevisualized under UV irradiation. In the case of the assay forthe stability of the PVIm-R/DNA complexes, gel electrophoresiswas run in the presence of dextran sulfate (1-20 mM as sulfategroup) incubated with each sample at room temperature for 10min.

Pyrene Fluorescence. A known amount of pyrene in acetonesolution was added to PBS (-), resulting in the solution at afinal concentration of 6.0 × 10-7 M. The pyrene solution (1mL) was mixed with the PVIm-R/DNA complexes in 1 mL ofPBS (-) where 29 µg/mL DNA was used at a +/- ratio of 1,4, or 12. The sample solution was incubated overnight at roomtemperature, and emission spectra with excitation at 337 nmwere recorded. The fluorescence intensity ratio of the first bandat 373 nm to the third band at 384 nm (I1/I3) was analyzed atpH 7.4 or pH 6.0.

Hemolysis Assay. The PVIm-R/DNA complexes were pre-pared at a +/- ratio of 12 with 10 mM sodium phosphate buffer(pH 7.4 or pH 6.0) containing 130 mM NaCl. Then, 150 µL ofthe resulting sample containing 186 µg of PVIm-R wasincubated with 20 µL of preserved sheep blood (Cosmo BioCo., Ltd., Tokyo, Japan) for 120 mim at 37 °C. After centri-fugation (13000 rpm, 1 min, 4 °C), the released hemoglobinwas determined by measuring the absorbance at 570 nm with aModel-550 microplate reader (Bio-Rad Laboratories, Inc.,Tokyo, Japan).

Cell Viability Assay. HepG2 cells (a gift from the JapanHealth Sciences Foundation), human hepatoma cell line, werecultured in tissue culture flasks containing Dulbecco’s modifiedEagle’s medium supplemented with 10% heat-inactivated FBS.The cells were seeded at 1 × 104 cells/well in a 96-well plateand incubated overnight at 37 °C in a 5% CO2 incubator. Thecells were treated with each polymer (0-400 µg/mL) andincubated for 24 h at 37 °C. By further incubation for 4 h, thecell viability was measured using the Alamar Blue assay (15)in triplicate.

Transfection Procedure. In a typical 96-well plate experi-ment, 1 × 104 cells/well HepG2 cells were transfected inDulbecco’s modified Eagle’s medium supplemented with 10%heat-inactivated FBS by the addition of 15 µL of PBS (-)containing 200 ng of plasmid DNA encoding the modified fireflyluciferase (pGL3-Control Vector; from Promega Co.) andcomplexed with polycations. After 1 day of incubation, themedium was removed, and the cells were further incubated for2 days in Dulbecco’s modified Eagle’s medium supplementedwith 10% FBS. Then, the cells were subjected to the luciferaseassay (Promega kit) according to the manufacturer’s instructions.Luciferase activities were normalized by protein concentrationsand are presented as relative light units (RLU). Proteinconcentrations were determined by the BCA protein assay kit(Pierce) according to the manufacturer’s instructions.

RESULTS AND DISCUSSION

Synthesis of PVIm-R. As shown in Scheme 1, PVIm (2)was reacted with an alkyl halide such as 1-bromobutane foralkylation to obtain quaternary imidazole groups. The number-average molecular weight of each resulting polymer (3)determined by GFC was about 8.8 × 103. The 1H NMRspectrum of the resulting polymers showed the characteristicsignals of both PVIm (16) [δ 1.8-2.2 (methylene), 2.3-3.7(methine), and 6.4-7.2 (imidazole) ppm] and alkyl [δ 0.7-0.8(terminal-methyl), 1.0-1.2 (3-methylene of butyl or 3,4,5,6,7-methylene of octyl), and 1.4-1.7 (2-methylene) ppm] moieties.From the signal ratio, the content (density) of alkylatedimidazole groups was calculated. Thus, we have synthesizedPVIm with quaternary imidazole groups, that is, PVIm-R.

pH-Dependent Behavior of PVIm-R in Water. To examinethe ionic properties of the remaining imidazole groups, we

Scheme 1. Synthesis of Alkylated Poly(1-vinylimidazole)

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carried out the acid-base titration of the resulting PVIm-Rsolution, as shown in Figure 1. Butylated PVIm, that is, PVIm-Bu, was chosen for representative PVIm-R. The imidazoleprotons of the PVIm-Bu were gently dissociated around pH 6so that the pKa of the PVIm-Bu was considered to be around 6.The proton dissociation profile approximately depended on thecontent of butylated imidazole groups. Namely, the 18 mol %butylated PVIm exhibited larger capacity of proton bufferingaround pH 6 because PVIm-Bu had more unmodified imidazolegroups with a pKa around 6. However, proton buffering of the40 mol % butylated PVIm occurred in a little lower region ofpH, suggesting that proton dissociation occurred easily becauseof the more hydrophobic environment near the imidazole groups.It should be noted that the aqueous solution of PVIm-Buexhibited no significant turbidity above pH 6 in spite of thedeprotonation of imidazole groups (Figure 1, inset). These resultssuggest that PVIm-R such as PVIm-Bu possessed a largecapacity of proton buffering at endosomal pH and that thePVIm-R was water-soluble despite the deprotonation of theimidazole groups.

pH-dependent Complex Formation between DNA andPVIm-R. We examined whether the PVIm-Bu, as representativePVIm-R, formed the polyion complexes with DNA by agarosegel electrophoresis (Figure 2A). At pH 7.4, no band wasobserved when the DNA was mixed with an excess of PVIm-Bu (lanes 4-6). The band disappearance is considered to beattributed to the induction of the coil-globule transition of DNAand the resulting inhibition of the intercalation of EtBr (17, 18).In particular, almost no free DNA was observed at the [butylatedimidazole]PVIm-Bu/[phosphate]DNA ratio of 1 (lane 3), where theamount of the cationic butylated imidazole groups of PVIm-Bu was equal to the anionic phosphate groups of DNA. Theexcess PVIm-Bu polymers are considered to remain in the freestate (Figure S-1, Supporting Information). Even in the presenceof an excess amount of the unmodified PVIm at pH 7.4, it isreported that most of the DNA migrated into the plus pole ofthe gel owing to the complete deprotonation of the imidazolegroups (13, 19). These results suggest that the PVIm-Bu formedthe DNA complexes at the stoichiometric charge ratio of DNAto butylated imidazole groups; that is, the butylated imidazolegroups worked as unique anchor groups to retain DNA.Furthermore, the free DNA observed at the [butylated imida-zole]PVIm-Bu/[phosphate]DNA ratio of 0.5 at pH 7.4 (lane 2)completely disappeared at pH 6.0 (lane 2′). This is due to theprotonation of the unmodified imidazole groups of the PVImbackbone.

To examine further the pH-dependent behavior of the DNAcomplexes with PVIm-Bu, we investigated the hydrophobicity

of the complexes by using the fluorescence of pyrene. Anemission intensity ratio of the first (373 nm) to the third (384nm) peaks of pyrene, I1/I3, is known to be sensitive to themicroenvironmental polarity surrounding the pyrene molecule(20). Consequently, this ratio has been widely used toestimate the hydrophobic nature (21-23). Namely, since thisparameter decreases with an increase of hydrophobicity, itrepresents hydrophilicity. Figure 2B depicts the I1/I3 ratio ofpyrene fluorescence in the buffer containing various PVIm-Bu/DNA complexes at pH 7.4 or pH 6.0. In buffers dissolvingDNA, the I1/I3 ratios of pyrene were approximately 1.6 at pH7.4 and pH 6.0. The presence of PVIm-Bu affected hydrophi-licity (I1/I3 ratio), which tended to decrease as the [butylatedimidazole]PVIm-Bu/[phosphate]DNA ratio increased. These resultssuggest that the PVIm-Bu/DNA complexes formed the domainwith a hydrophobic nature. Furthermore, it should be noted thathydrophilicity (I1/I3 ratio) in the presence of the excess amountof PVIm-Bu ([butylated imidazole]PVIm-Bu/[phosphate]DNA ) 4or 12) at pH 6.0 was lower than that at pH 7.4. The resultinghydrophobic nature is therefore considered to increase even afterthe protonation of imidazole groups of the PVIm-Bu. Theseresults suggest that the protonated PVIm-Bu enhanced themicelle formation which consisted of the shell of the protonatedimidazole groups and the core of the butylated imidazole groups.It can be said that the PVIm-Bu/DNA complexes were capableof varying their hydrophobic-hydrophilic balance in responseto endosomal pH.

Biochemical Properties of the PVIm-R/DNA Complexes.To examine the effect of the resulting pH-dependent change ofthe hydrophobic-hydrophilic balance on the interaction withthe real cell membranes, we measured the hemolytic activity

Figure 1. Acid-base titration curves of PVIm-R: (b) 18 mol %butylated PVIm; (O) 40 mol % butylated PVIm. Acidic polymersolutions (3.3 mg/mL) were titrated with the stepwise addition of 0.2M NaOH. (Inset) Effect of pH on the solubility of the PVIm-R in water(3.3 mg/mL): (b) 18 mol % butylated PVIm; (O) 40 mol % butylatedPVIm. The turbidity was measured by monitoring the absorbance at500 nm of the polymer aqueous solution during acid-base titration.

Figure 2. (A) Analysis of the pH-dependent formation of the complexesbetween DNA and PVIm-R by agarose gel electrophoresis. Interactionof 18 mol % butylated PVIm with DNA at pH 7.4 (lanes 1-6) or pH6.0 (lanes 1′-6′): lanes 1 and 1′, DNA alone; lanes 2-6 and 2′-6′,PVIm-Bu/DNA mixtures at different unit ratios relative to butylatedimidazole groups of PVIm-Bu per phosphate group of DNA ([butylatedimidazole]/[phosphate] ) 0.5, 1, 2, 6, or 12), lanes 2 and 2′, 0.5; lanes3 and 3′, 1; lanes 4 and 4′, 2; lanes 5 and 5′, 6; lanes 6 and 6′, 12. Thesolid arrowhead indicates the well where each sample was loaded. (B)I1/I3 of pyrene fluorescence in PBS(-) with PVIm-Bu/DNA complexesat pH 7.4 (O) or pH 6.0 (b). The complexes were formed at +/- ratioof 1, 4, or 12 where the final concentration of DNA was 14.5 µg/mL.I1/I3 was defined as the fluorescence intensity ratio of the first band at373 nm to the third band at 384 nm where the final concentration ofpyrene was 0.3 µM.

648 Bioconjugate Chem., Vol. 21, No. 4, 2010 Asayama et al.

of the PVIm-Bu/DNA complexes as representative PVIm-R/DNA complexes (Figure 3, inset). The complexes causednegligible hemolysis at pH 7.4, whereas the hemolytic activitysignificantly increased at pH 6.0. These results suggest that themembrane disruptive activity of the PVIm-Bu/DNA complexesincreased at endosomal pH. The hydrophobic-hydrophilicbalance of the PVIm-Bu/DNA complexes at endosomal pH isconsidered to enhance membrane disruptive activity. It istherefore expected that PVIm-Bu enhances the ability to escapefrom acidic endosomal vesicles.

As a result of the membrane disruptive activity at endosomalpH, we first examined the gene expression mediated by thePVIm-Bu/DNA complexes. As shown in Figure 3, the PVIm-Bu/DNA complexes mediated remarkable gene expression,which was higher than that mediated by the control PEI. Itshould be noted that the PVIm-Bu/DNA complexes showedgene expression values approximately 100 times higher thanthat of the PVIm-NH2/DNA complexes. These results suggestthat not PVIm-NH2 but PVIm-Bu possessed the requiredproperties as a gene carrier. A main property is considered tobe the stabilization of the DNA complexes, not the protonbuffering, by the introduced alkyl groups such as butyl groups.

Stability of PVIm-R/DNA Complexes. To confirm thestability of the PVIm-R/DNA complexes, we attempted torelease DNA from the polyion complexes by competitiveexchange with other polyanions (24). For effective transfection,the release of DNA should not happen outside the target cell,whereas that must occur somewhere inside to allow the bindingof the transcription machinery. In biological fluids, the proteinsborne by various anionic polysaccharides circulate. As anextreme case, dextran sulfate was used as a polyanion; namely,the agarose gel electrophoresis was carried out after the PVIm-R/DNA complexes were incubated with dextran sulfates. Theresults are shown in Figure 4. As the concentration of the dextransulfate increased, the DNA increasingly migrated (lanes 4-6)

in the case of PVIm-NH2/DNA complexes. However, in caseof DNA complexes with octylated PVIm, that is, PVIm-Oc,almost no DNA migrated even in the presence of a higherconcentration of dextran sulfate (lane 6′′). These results suggestthat PVIm-NH2/DNA complexes easily released DNA byexposure to polyanions and that PVIm-Oc/DNA complexesstably retained DNA. It is worth noting that it was hard tomigrate the DNA in the case of the PVIm-Bu/DNA complexes,as compared with the PVIm-NH2/DNA complexes (lane 4′). Thisis probably caused by an adequate length of alkyl chains tostabilize the electrostatic interaction between PVIm-R and DNA.It is reported that the stability of polycation/DNA complexesdepends on the chain length of a whole polymer; namely, thelonger polycation is found to interact with DNA more stronglythan the shorter one (25-27). In this study, the stability ofpolycation/DNA complexes has depended on the chain lengthof introduced alkyl chains in the polycation, which promises aunique design of polycation/DNA complexes. We have thereforeconsidered that PVIm-Bu/DNA complexes have the ability toretain DNA stably outside the target cell and to release DNAadequately inside the cell for transcription.

Cytotoxicity of PVIm-R/DNA Complexes. Cytotoxicity ofa gene carrier is an important factor for clinical applications.Free polycations exist solely when DNA is released from thepolyion complexes (28). Furthermore, the overall cytotoxicityof free polycations is higher than that of the correspondingcomplexes. Accordingly, we chose the cytotoxicity assay of thefree polycations to give a worst case estimating the interactionof the polycations with cells rather than that of the polyioncomplexes with DNA. As shown in Figure 5, we thereforeexamine the effect of PVIm-R on cell viability. The viabilityof HepG2 hepatoma cells did not significantly decrease whenPVIm-Bu was added up to the concentration of 400 µg/mL,which was higher than the transfection conditions. Consequently,it is worth noting that PVIm-Bu exhibited no apparent cyto-toxicity. The control methylated and ethylated PVIm, that is,PVIm-Me and PVIm-Et, respectively, exhibited the sametendency as PVIm-Bu. However, little viability was observedwhen PVIm-Oc, as well as the control PEI, was added up to 50

Figure 3. Transfection of luciferase gene to HepG2 cells by the DNAcomplexes with PVIm-R. As PVIm-R, 18 mol % butylated PVIm wasused at the +/- ratio of 12 or 16. The PVIm-NH2/DNA complexes at+/- ratio of 12 and the PEI/DNA complexes at +/- ratio of 12 or 16were used as the control. The cells (1 × 104 cells/well) were transfectedby adding 200 ng of plasmid DNA complexed with polycations for 1day in the presence of 10% FBS. Gene expression was determined 2days later as RLU normalized by protein concentrations. Symbols anderror bars represent the mean and standard deviation of the measure-ments made in paired samples (n ) 3). (Inset) Effect of pH on thehemolytic activity of PVIm-R/DNA complexes. Erythrocytes wereincubated with the PVIm-Bu/DNA complexes at the +/- ratio of 12for 120 min at 37 °C in 10 mM sodium phosphate buffer (pH 7.4 orpH 6.0) containing 130 mM NaCl. The released hemoglobin wasdetermined by measuring the absorbance at 570 nm (Abs570), wherethe Abs570 in the absence of the PVIm-Bu/DNA complexes (pH 7.4 orpH 6.0) was used as a baseline. Symbols and error bars represent themean and standard deviation of the measurements made in pairedsamples (n ) 3). * indicates statistical significance (p < 0.02) whencompared to the pH 7.4 value.

Figure 4. Release of DNA from PVIm-R/DNA complexes by dextransulfates (DS) as assessed by agarose gel electrophoresis: lanes 1, 1′,and 1′′, DNA alone; lanes 2, 2′, and 2′′, 0 mM; lanes 3, 3′, and 3′′, 1mM; lanes 4, 4′, and 4′′, 5 mM; lanes 5, 5′, and 5′′, 10 mM; lanes 6,6′, and 6′′, 20 mM. The DNA mixtures with 18 mol % butylated PVIm(PVIm-Bu) or 18 mol % octylated PVIm (PVIm-Oc) at the +/- ratioof 12 were incubated for 10 min at room temperature in the presence(lanes 3′-6′ and 3′′-6′′) or absence (lanes 2′ and 2′′) of DS (1-20mM as sulfate group), followed by loading to the gel. As the control,the DNA mixture with PVIm-NH2 at the +/- ratio of 12 was used inthe presence (lanes 3-6) or absence (lane 2) of DS. The solid arrowheadindicates the well where each sample was loaded.

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µg/mL. These results suggest that PVIm-R with a relatively shortlength of alkyl groups such as butyl, ethyl, or methyl groupspromises to be a noncytotoxic gene carrier. The noncytotoxicproperty may be attributed to the partial shielding of surfacecharge, as poly(ethylene glycol) (29), by unmodified imidazolegroups. However, PVIm-R with a relatively long length of alkylgroups such as octyl groups is considered to cause significantcytotoxicity by the presence of the more amphiphilic propertyfor cell membrane damage. Actually, a little significant cyto-toxicity was observed when PVIm-Bu was added up to theconcentration of 1000 µg/mL, where PVIm-Me and PVIm-Etexhibited no significant cytotoxicity (Figure S-2, SupportingInformation).

Gene Delivery by PVIm-R/DNA Complexes. As a resultof no apparent cytotoxicity, we further examined the geneexpression mediated by PVIm-R/DNA complexes in view ofthe density of alkylated imidazole groups. As shown in Figure6, the PVIm-Bu polycations with different densities of butylatedimidazole groups were used as representative PVIm-R for DNAcomplex formation. Little gene expression was observed at any+/- ratio when we used the DNA complexes with PVIm-Buwith a lower density (7 mol %) of butylated imidazole groups.In case of the PVIm-Bu with a higher density (38 mol %) ofbutylated imidazole groups, however, the DNA complexesmediated remarkable gene expression even if the complexes atthe +/- ratio of 4 was used. Although the DNA complexeswith PVIm-Bu with a middle density (23 mol %) of butylatedimidazole groups mediated little gene expression at the +/-ratio of 4, the DNA complexes at higher +/- ratios succeeded

in remarkable gene expression. These results suggest that thedensity of the butylated imidazole groups in PVIm-Bu was animportant factor for gene delivery.

As a result of the dependency of the density of the butylatedimidazole groups, we finally examined the gene expressionmediated by the PVIm-R/DNA complexes in view of the alkylchain length of alkylated imidazole groups. Figure 7 shows theeffect of the alkyl chain length of PVIm-R with a middle density(20 mol %) of alkylated imidazole groups on gene expression.As expected, PVIm-Oc mediated little gene expression becauseof too stable retention of DNA (Figure 4) and significantcytotoxicity (Figure 5). As a result of PVIm-Bu with a middledensity (18 mol %, Figure 3; 23 mol %, Figure 6) of alkylatedimidazole groups, the gene expression mediated by the PVIm-Bu/DNA complexes depended on the +/- ratio. It should benoted that PVIm-Me and PVIm-Et mediated higher geneexpression than PVIm-Bu at lower +/- ratios. Especially, thegene expression mediated by the PVIm-Et/DNA complexes didnot depend on the +/- ratio. These results suggest that the alkylchain length of the alkylated imidazole groups was also animportant factor for gene delivery.

Gene expression decreased with a decrease in the density ofthe alkylated (butylated) imidazole groups (Figure 6). The highergene expression mediated by PVIm-Me and PVIm-Et istherefore unexpected (Figure 7) because the shorter length ofthe alkyl chain decreased the apparent density of the alkylatedimidazole groups. However, it is surprising that the PVIm-Bu/DNA complex with a middle density of butylated imidazolegroups was more stable than that with higher density (FigureS-3, Supporting Information). The excess butylated imidazolegroups are therefore considered to enhance the membranedisruptive activity to escape from acidic endosomal vesicles.Furthermore, it is also surprising that the DNA complex withPVIm-Me or PVIm-Et was more stable than that with PVIm-Bu (Figure S-4, Supporting Information). At present, the stabilitymechanism is unclear; analysis is now in progress for moredetailed investigations. Nevertheless, it can be said that thePVIm-Et/DNA complex as well as the PVIm-Me complex hasmore adequate retention of DNA for gene delivery, as comparedwith the PVIm-Bu/DNA complex. Although amphiphilic im-idazolinium compounds for gene delivery were previouslyreported, the compounds form liposomes whose stabilitydepends on the length of the dialkyl groups of imidazolinium(30). In this study, many alkyl groups in PVIm-R are consideredto be intertwined with DNA grooves and the degree ofintertwining may affect the transcription of the delivered gene.

Figure 5. Effect of PVIm-R on the viability of HepG2 cells after 1day of incubation: (b) 20 mol % butylated PVIm (PVIm-Bu); (9) 20mol % octylated PVIm (PVIm-Oc); (O) 20 mol % ethylated PVIm(PVIm-Et); (0) 20 mol % methylated PVIm (PVIm-Me); (2) PEI.Symbols and error bars represent the mean and standard deviation ofthe measurements made in triplicate wells.

Figure 6. Effect of the density of the alkylated imidazole groups onthe transfection activity mediated by PVIm-R/DNA complexes. AsPVIm-R, 7 mol % butylated PVIm (white bars), 23 mol % butylatedPVIm (gray bars), or 38 mol % butylated PVIm (black bars) was usedfor the transfection. The PVIm-Bu/DNA complexes were prepared atthe +/- ratio of 4, 8, 12, 24, or 36. Other experimental conditions arethe same as those described in Figure 3.

Figure 7. Effect of the length of the alkylated imidazole groups on thetransfection activity mediated by PVIm-R/DNA complexes. As PVIm-R, 20 mol % methylated PVIm (PVIm-Me) (white bars), 20 mol %ethylated PVIm (PVIm-Et) (gray bars), 20 mol % butylated PVIm(PVIm-Bu) (black bars), or 20 mol % octylated PVIm (PVIm-Oc)(hatched bars) were used for the transfection. The PVIm-R/DNAcomplexes were prepared at the +/- ratio of 4, 12, or 36. Otherexperimental conditions are the same those described in Figure 3.

650 Bioconjugate Chem., Vol. 21, No. 4, 2010 Asayama et al.

Taking these results into account, alkylated imidazole groupsare essential and unique functional groups in PVIm-R forefficient gene delivery.

Conclusions. We have synthesized a unique pH-sensitivepolycation PVIm-R and evaluated the physicochemical andbiochemical properties of the pH-sensitive gene carrier. Theresulting PVIm-R was water-soluble in spite of the deprotonationof the imidazole groups at physiological pH. PVIm-R/DNAcomplex formation was mediated by alkylated imidazole groupsworking as anchor groups to retain DNA. The DNA complexwith butylated PVIm (PVIm-Bu) formed the domain with ahydrophobic nature, which increased after the protonation ofimidazole groups of PVIm-Bu. The increased hydrophobic-hydrophilic incline of the PVIm-R/DNA complexes at endo-somal pH enhanced membrane disruptive activity. PVIm-Buexhibited no significant cytotoxicity in spite of the existence ofcationic groups and easily released DNA, as compared withPVIm-Oc. By using PVIm-R as a pH-sensitive DNA carrier,PVIm-R/DNA complexes mediated efficient gene deliveryattributed to the alkylated imidazole groups, and gene expressiondepended on the length and density of the alkyl chains.Consequently, PVIm-R’s control of the stability of DNA polyioncomplexes enhanced noncytotoxic gene delivery by optimizedalkylated imidazole groups. The control of the polycation/DNAcomplex properties by varying the length and density of thealkyl chains grafted onto PVIm is expected to offer uniquedesigns for gene delivery systems.

Supporting Information Available: Analysis of the forma-tion of the complex between DNA and PVIm-Bu by agarosegel electrophoresis in the absence of EtBr; effect of PVIm-Ron the viability of HepG2 cells after 1 day incubation; releaseof DNA from PVIm-Bu/DNA complexes by dextran sulfates(DS) as assessed by agarose gel electrophoresis; and releaseof DNA from PVIm-R/DNA complexes by dextran sulfates(DS) as assessed by agarose gel electrophoresis. This materialis available free of charge via the Internet at http://pubs.acs.org.

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