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Bioconjugate Chem. 2010, 21, 646–652
Synthesis and Characterization of Alkylated Poly(1-vinylimidazole) to Control 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 polycation to 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-base titration and solution turbidity measurements. Agarose gel retardation assay proved that the alkylated imidazole groups worked as anchor groups to retain DNA. Pyrene fluorescence measurement showed that the hydrophobic domain of the DNA complex with butylated PVIm (PVIm-Bu) increased after the protonation of imidazole groups of the PVIm-Bu to enhance the membrane disruptive activity. The PVIm-Bu exhibited no significant cytotoxicity in spite of the existence of cationic groups. The resulting PVIm-Bu/DNA complexes easily released DNA, as compared with the octylated PVIm, which was examined by competitive exchange with dextran sulfate. As a result, the PVIm-R/DNA complexes mediated efficient gene delivery, and the gene expression depended on the length and density of the alkyl chains. These results suggest that pH-sensitive PVIm-R’s control of the stability of DNA polyion complexes enhanced noncytotoxic gene delivery by the optimized alkylated imidazole groups.
INTRODUCTION In gene delivery systems, the formation of polycation/DNA polyion complexes is a key factor for the new design of efficient delivery (1-3). The polyion complexes on the cell plasma membrane are internalized into acidic endosomal vesicles where they are subjected to a significant pH change from pH 7 to 5 (4). Endosomal escape is one of the critical factors for efficient gene 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 delivered material from endosomes (5-7). PEI induces swelling of the endosomes that leads to membrane disruption, that is, the proton sponge effect. Recently, polymers modified with histidine or other moieties containing an imidazole group have shown significant enhancement of gene expression without increasing cytotoxicity compared with that of nonmodified polymers (8-12). In this case, histidine or other moieties containing an imidazole group have made polycation/DNA complexes escape from an endosome by a proton sponge mechanism. The imidazole heterocycles displaying a pKa around 6 possess buffering capacity in endosomal pH, inducing membrane destabilization after their protonation. The resulting imidazole groups facilitate the release of 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 cellspecific gene delivery (13). By using PVIm-NH2 as a pH* To whom correspondence should be addressed. Tel: +81-42-6771111 (ext.) 4976. Fax: +81-42-677-2821. E-mail: asayama-shoichiro@ c.metro-u.ac.jp. 1 Abbreviations: PEI, poly(ethylenimine); PVIm, poly(1-vinylimidazole); PVIm-NH2, aminated poly(1-vinylimidazole); PVIm-R, alkylated 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 light unit; DS, dextran sulfate.
sensitive DNA carrier, as well as a lactosylated poly(L-lysine) as a cell-targeting DNA carrier, the resulting ternary complexes specifically mediate gene expression. Gene expression depends on our new concept that DNA ternary complexes dissociate ligand polycations in response to endosomal pH (13, 14). However, PVIm-NH2/DNA binary complexes mediate no significant gene expression. In this study, to develop PVIm-NH2 for the realization of efficient gene expression, we have synthesized PVIm with several alkylated imidazole groups, that is, alkylated PVIm (PVIm-R). PVIm is a water-soluble homopolymer possessing many imidazole groups. In spite of a large capacity for H+ buffering at endosomal pH, PVIm has difficulty in forming complexes with DNA at physiological pH because its imidazole groups are negligibly charged at physiological pH. The introduced alkylated imidazole groups with a quaternary nitrogen atom are expected to work as new anchor groups to retain DNA and to control the stability of polyion complexes. The control of the stability of the polyion complexes by the length and density of the alkylated imidazole groups has no precedent, to the best of our knowledge. Consequently, pH-sensitive PVImR’s control of the stability of DNA polyion complexes is expected to mediate efficient gene expression by optimizing the length and density of the alkylated imidazole groups.
EXPERIMENTAL PROCEDURES Materials. 1-Vinylimidazole (VIm), 1-bromobutane, 1-bromooctane, and pyrene were purchased from Aldrich Chemical Co. (Milwaukee, WI). VIm was distilled under reduced pressure. 2,2′-Azobis(2,4-dimethylvaleronitrile) (V-65) and bromoethane were 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 testis deoxyribonucleic acid (DNA) sodium salt were from Sigma Chemical Co. (St. Louis, MO). All other chemicals of a special grade were used without further purification.
10.1021/bc900411m 2010 American Chemical Society Published on Web 04/05/2010
Alkylated Poly(1-vinylimidazole) Scheme 1. Synthesis of Alkylated Poly(1-vinylimidazole)
Synthesis of PVIm-R. The synthetic route of PVIm-R is shown in Scheme 1. VIm (1) (300 µL) and V-65 (15.8 mg) as an initiator were dissolved in 2.7 mL of N,N-dimethylformamide (DMF). The radical polymerization reaction was carried out at 45 °C for 1 day. After the reaction, the content was poured into a large excess of acetone, and the precipitate was dried in Vacuo. The resulting polymer (2) (25 mg) and various amounts of alkyl halide (iodomethane, bromoethane, 1-bromobutane, or 1-bromooctane) (3-100 µL) were dissolved in 2 mL of DMF. The reaction was carried out at 40 °C for 1-6 days according to each case. The reaction mixture was poured into a large excess of diethyl ether. The precipitate was dried in Vacuo and dissolved in water. After dialysis against distilled water using a Spectra/ Por 7 membrane (molecular weight cutoff ) 103), the resulting polymer (3) was obtained by freeze-drying. Gel Filtration Chromatography (GFC). GFC was carried out using a JASCO PU-980 pumping system (Tokyo, Japan) at the flow rate of 1.0 mL/min with a Shodex OHpak SB-804 HQ column (Showa Denko K. K., Tokyo, Japan). The aqueous solution containing 0.5 M CH3COOH and 0.2 M NaNO3 was used as a mobile phase. One hundred microliters of 1 mg/mL samples were injected into the column. Eluate was detected by a refractive index detector (RI-1530, JASCO). Calibration was made with polyethylene glycol standards. 1 H NMR Spectroscopy. Each polymer (3 mg) was dissolved in 700 µL of D2O (99.8 atom % deuterium; Acros, NJ). The 1H NMR spectra (400 MHz) were obtained by a JEOL JNM-AL400 spectrometer (Tokyo, Japan). Acid-Base Titration and Turbidity Measurement of PVIm-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 acidic polymer solution (pH 4) was titrated with a 0.2 M NaOH solution. The pH value was checked with a pH meter (model F-52T, Horiba, Kyoto, Japan). The titration was carried out by the stepwise addition of 0.2 M NaOH and stopped at pH 10. The turbidity of the solution during the titration was measured by monitoring the absorbance at 500 nm with a spectrophotometer (model Ubest-55, JASCO, Tokyo, Japan). Agarose Gel Retardation Assay. Salmon testis DNA was dissolved in PBS (-) at 1.1 mg/mL. The resulting DNA stock solution was added to the polymer solutions in 50 mM sodium phosphate buffer (pH 7.5 or pH 6.0) at various polymer/DNA ratios. The final diluted concentration of DNA was adjusted to 66.7 µg/mL. After 30 min of incubation at room temperature, each sample (corresponding to 1 µg of DNA) was mixed with a loading buffer and loaded onto a 1% agarose gel containing 1 µg/mL of ethidium bromide (EtBr). Gel electrophoresis was run at room temperature in 50 mM sodium phosphate buffer
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(pH 7.5 or pH 6.0) at 50 V for 15 min. The DNA bands were visualized under UV irradiation. In the case of the assay for the stability of the PVIm-R/DNA complexes, gel electrophoresis was run in the presence of dextran sulfate (1-20 mM as sulfate group) incubated with each sample at room temperature for 10 min. Pyrene Fluorescence. A known amount of pyrene in acetone solution was added to PBS (-), resulting in the solution at a final concentration of 6.0 × 10-7 M. The pyrene solution (1 mL) was mixed with the PVIm-R/DNA complexes in 1 mL of PBS (-) where 29 µg/mL DNA was used at a +/- ratio of 1, 4, or 12. The sample solution was incubated overnight at room temperature, and emission spectra with excitation at 337 nm were recorded. The fluorescence intensity ratio of the first band at 373 nm to the third band at 384 nm (I1/I3) was analyzed at pH 7.4 or pH 6.0. Hemolysis Assay. The PVIm-R/DNA complexes were prepared 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 of the resulting sample containing 186 µg of PVIm-R was incubated with 20 µL of preserved sheep blood (Cosmo Bio Co., Ltd., Tokyo, Japan) for 120 mim at 37 °C. After centrifugation (13000 rpm, 1 min, 4 °C), the released hemoglobin was determined by measuring the absorbance at 570 nm with a Model-550 microplate reader (Bio-Rad Laboratories, Inc., Tokyo, Japan). Cell Viability Assay. HepG2 cells (a gift from the Japan Health Sciences Foundation), human hepatoma cell line, were cultured in tissue culture flasks containing Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated FBS. The cells were seeded at 1 × 104 cells/well in a 96-well plate and incubated overnight at 37 °C in a 5% CO2 incubator. The cells were treated with each polymer (0-400 µg/mL) and incubated for 24 h at 37 °C. By further incubation for 4 h, the cell viability was measured using the Alamar Blue assay (15) in triplicate. Transfection Procedure. In a typical 96-well plate experiment, 1 × 104 cells/well HepG2 cells were transfected in Dulbecco’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 firefly luciferase (pGL3-Control Vector; from Promega Co.) and complexed with polycations. After 1 day of incubation, the medium was removed, and the cells were further incubated for 2 days in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. Then, the cells were subjected to the luciferase assay (Promega kit) according to the manufacturer’s instructions. Luciferase activities were normalized by protein concentrations and are presented as relative light units (RLU). Protein concentrations 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 for alkylation to obtain quaternary imidazole groups. The numberaverage molecular weight of each resulting polymer (3) determined by GFC was about 8.8 × 103. The 1H NMR spectrum of the resulting polymers showed the characteristic signals 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 3,4,5,6,7methylene of octyl), and 1.4-1.7 (2-methylene) ppm] moieties. From the signal ratio, the content (density) of alkylated imidazole groups was calculated. Thus, we have synthesized PVIm with quaternary imidazole groups, that is, PVIm-R. pH-Dependent Behavior of PVIm-R in Water. To examine the ionic properties of the remaining imidazole groups, we
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Figure 1. Acid-base titration curves of PVIm-R: (b) 18 mol % butylated PVIm; (O) 40 mol % butylated PVIm. Acidic polymer solutions (3.3 mg/mL) were titrated with the stepwise addition of 0.2 M 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 % butylated PVIm. The turbidity was measured by monitoring the absorbance at 500 nm of the polymer aqueous solution during acid-base titration.
carried out the acid-base titration of the resulting PVIm-R solution, as shown in Figure 1. Butylated PVIm, that is, PVImBu, was chosen for representative PVIm-R. The imidazole protons of the PVIm-Bu were gently dissociated around pH 6 so that the pKa of the PVIm-Bu was considered to be around 6. The proton dissociation profile approximately depended on the content of butylated imidazole groups. Namely, the 18 mol % butylated PVIm exhibited larger capacity of proton buffering around pH 6 because PVIm-Bu had more unmodified imidazole groups with a pKa around 6. However, proton buffering of the 40 mol % butylated PVIm occurred in a little lower region of pH, suggesting that proton dissociation occurred easily because of the more hydrophobic environment near the imidazole groups. It should be noted that the aqueous solution of PVIm-Bu exhibited no significant turbidity above pH 6 in spite of the deprotonation of imidazole groups (Figure 1, inset). These results suggest that PVIm-R such as PVIm-Bu possessed a large capacity of proton buffering at endosomal pH and that the PVIm-R was water-soluble despite the deprotonation of the imidazole groups. pH-dependent Complex Formation between DNA and PVIm-R. We examined whether the PVIm-Bu, as representative PVIm-R, formed the polyion complexes with DNA by agarose gel electrophoresis (Figure 2A). At pH 7.4, no band was observed when the DNA was mixed with an excess of PVImBu (lanes 4-6). The band disappearance is considered to be attributed to the induction of the coil-globule transition of DNA and the resulting inhibition of the intercalation of EtBr (17, 18). In particular, almost no free DNA was observed at the [butylated imidazole]PVIm-Bu/[phosphate]DNA ratio of 1 (lane 3), where the amount of the cationic butylated imidazole groups of PVImBu was equal to the anionic phosphate groups of DNA. The excess PVIm-Bu polymers are considered to remain in the free state (Figure S-1, Supporting Information). Even in the presence of an excess amount of the unmodified PVIm at pH 7.4, it is reported that most of the DNA migrated into the plus pole of the gel owing to the complete deprotonation of the imidazole groups (13, 19). These results suggest that the PVIm-Bu formed the DNA complexes at the stoichiometric charge ratio of DNA to butylated imidazole groups; that is, the butylated imidazole groups worked as unique anchor groups to retain DNA. Furthermore, the free DNA observed at the [butylated imidazole]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 the protonation of the unmodified imidazole groups of the PVIm backbone. To examine further the pH-dependent behavior of the DNA complexes with PVIm-Bu, we investigated the hydrophobicity
Figure 2. (A) Analysis of the pH-dependent formation of the complexes between DNA and PVIm-R by agarose gel electrophoresis. Interaction of 18 mol % butylated PVIm with DNA at pH 7.4 (lanes 1-6) or pH 6.0 (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 butylated imidazole groups of PVIm-Bu per phosphate group of DNA ([butylated imidazole]/[phosphate] ) 0.5, 1, 2, 6, or 12), lanes 2 and 2′, 0.5; lanes 3 and 3′, 1; lanes 4 and 4′, 2; lanes 5 and 5′, 6; lanes 6 and 6′, 12. The solid arrowhead indicates the well where each sample was loaded. (B) I1/I3 of pyrene fluorescence in PBS(-) with PVIm-Bu/DNA complexes at pH 7.4 (O) or pH 6.0 (b). The complexes were formed at +/- ratio of 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 at 373 nm to the third band at 384 nm where the final concentration of pyrene was 0.3 µM.
of the complexes by using the fluorescence of pyrene. An emission intensity ratio of the first (373 nm) to the third (384 nm) peaks of pyrene, I1/I3, is known to be sensitive to the microenvironmental polarity surrounding the pyrene molecule (20). Consequently, this ratio has been widely used to estimate the hydrophobic nature (21-23). Namely, since this parameter decreases with an increase of hydrophobicity, it represents hydrophilicity. Figure 2B depicts the I1/I3 ratio of pyrene fluorescence in the buffer containing various PVIm-Bu/ DNA complexes at pH 7.4 or pH 6.0. In buffers dissolving DNA, the I1/I3 ratios of pyrene were approximately 1.6 at pH 7.4 and pH 6.0. The presence of PVIm-Bu affected hydrophilicity (I1/I3 ratio), which tended to decrease as the [butylated imidazole]PVIm-Bu/[phosphate]DNA ratio increased. These results suggest that the PVIm-Bu/DNA complexes formed the domain with a hydrophobic nature. Furthermore, it should be noted that hydrophilicity (I1/I3 ratio) in the presence of the excess amount of PVIm-Bu ([butylated imidazole]PVIm-Bu/[phosphate]DNA ) 4 or 12) at pH 6.0 was lower than that at pH 7.4. The resulting hydrophobic nature is therefore considered to increase even after the protonation of imidazole groups of the PVIm-Bu. These results suggest that the protonated PVIm-Bu enhanced the micelle formation which consisted of the shell of the protonated imidazole groups and the core of the butylated imidazole groups. It can be said that the PVIm-Bu/DNA complexes were capable of varying their hydrophobic-hydrophilic balance in response to endosomal pH. Biochemical Properties of the PVIm-R/DNA Complexes. To examine the effect of the resulting pH-dependent change of the hydrophobic-hydrophilic balance on the interaction with the real cell membranes, we measured the hemolytic activity
Alkylated Poly(1-vinylimidazole)
Figure 3. Transfection of luciferase gene to HepG2 cells by the DNA complexes with PVIm-R. As PVIm-R, 18 mol % butylated PVIm was used 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 16 were used as the control. The cells (1 × 104 cells/well) were transfected by adding 200 ng of plasmid DNA complexed with polycations for 1 day in the presence of 10% FBS. Gene expression was determined 2 days later as RLU normalized by protein concentrations. Symbols and error bars represent the mean and standard deviation of the measurements made in paired samples (n ) 3). (Inset) Effect of pH on the hemolytic activity of PVIm-R/DNA complexes. Erythrocytes were incubated with the PVIm-Bu/DNA complexes at the +/- ratio of 12 for 120 min at 37 °C in 10 mM sodium phosphate buffer (pH 7.4 or pH 6.0) containing 130 mM NaCl. The released hemoglobin was determined by measuring the absorbance at 570 nm (Abs570), where the Abs570 in the absence of the PVIm-Bu/DNA complexes (pH 7.4 or pH 6.0) was used as a baseline. Symbols and error bars represent the mean and standard deviation of the measurements made in paired samples (n ) 3). * indicates statistical significance (p < 0.02) when compared to the pH 7.4 value.
of the PVIm-Bu/DNA complexes as representative PVIm-R/ DNA complexes (Figure 3, inset). The complexes caused negligible hemolysis at pH 7.4, whereas the hemolytic activity significantly increased at pH 6.0. These results suggest that the membrane disruptive activity of the PVIm-Bu/DNA complexes increased at endosomal pH. The hydrophobic-hydrophilic balance of the PVIm-Bu/DNA complexes at endosomal pH is considered to enhance membrane disruptive activity. It is therefore expected that PVIm-Bu enhances the ability to escape from acidic endosomal vesicles. As a result of the membrane disruptive activity at endosomal pH, we first examined the gene expression mediated by the PVIm-Bu/DNA complexes. As shown in Figure 3, the PVImBu/DNA complexes mediated remarkable gene expression, which was higher than that mediated by the control PEI. It should be noted that the PVIm-Bu/DNA complexes showed gene expression values approximately 100 times higher than that of the PVIm-NH2/DNA complexes. These results suggest that not PVIm-NH2 but PVIm-Bu possessed the required properties as a gene carrier. A main property is considered to be the stabilization of the DNA complexes, not the proton buffering, by the introduced alkyl groups such as butyl groups. Stability of PVIm-R/DNA Complexes. To confirm the stability of the PVIm-R/DNA complexes, we attempted to release DNA from the polyion complexes by competitive exchange 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 binding of the transcription machinery. In biological fluids, the proteins borne by various anionic polysaccharides circulate. As an extreme case, dextran sulfate was used as a polyanion; namely, the agarose gel electrophoresis was carried out after the PVImR/DNA complexes were incubated with dextran sulfates. The results are shown in Figure 4. As the concentration of the dextran sulfate increased, the DNA increasingly migrated (lanes 4-6)
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Figure 4. Release of DNA from PVIm-R/DNA complexes by dextran sulfates (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′′, 1 mM; 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 +/- ratio of 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 (1-20 mM 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 in the presence (lanes 3-6) or absence (lane 2) of DS. The solid arrowhead indicates the well where each sample was loaded.
in the case of PVIm-NH2/DNA complexes. However, in case of DNA complexes with octylated PVIm, that is, PVIm-Oc, almost no DNA migrated even in the presence of a higher concentration of dextran sulfate (lane 6′′). These results suggest that PVIm-NH2/DNA complexes easily released DNA by exposure to polyanions and that PVIm-Oc/DNA complexes stably retained DNA. It is worth noting that it was hard to migrate the DNA in the case of the PVIm-Bu/DNA complexes, as compared with the PVIm-NH2/DNA complexes (lane 4′). This is probably caused by an adequate length of alkyl chains to stabilize the electrostatic interaction between PVIm-R and DNA. It is reported that the stability of polycation/DNA complexes depends on the chain length of a whole polymer; namely, the longer polycation is found to interact with DNA more strongly than the shorter one (25-27). In this study, the stability of polycation/DNA complexes has depended on the chain length of introduced alkyl chains in the polycation, which promises a unique design of polycation/DNA complexes. We have therefore considered that PVIm-Bu/DNA complexes have the ability to retain DNA stably outside the target cell and to release DNA adequately inside the cell for transcription. Cytotoxicity of PVIm-R/DNA Complexes. Cytotoxicity of a gene carrier is an important factor for clinical applications. Free polycations exist solely when DNA is released from the polyion complexes (28). Furthermore, the overall cytotoxicity of free polycations is higher than that of the corresponding complexes. Accordingly, we chose the cytotoxicity assay of the free polycations to give a worst case estimating the interaction of the polycations with cells rather than that of the polyion complexes with DNA. As shown in Figure 5, we therefore examine the effect of PVIm-R on cell viability. The viability of HepG2 hepatoma cells did not significantly decrease when PVIm-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 cytotoxicity. The control methylated and ethylated PVIm, that is, PVIm-Me and PVIm-Et, respectively, exhibited the same tendency as PVIm-Bu. However, little viability was observed when PVIm-Oc, as well as the control PEI, was added up to 50
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Figure 5. Effect of PVIm-R on the viability of HepG2 cells after 1 day of incubation: (b) 20 mol % butylated PVIm (PVIm-Bu); (9) 20 mol % 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 of the measurements made in triplicate wells.
Figure 6. Effect of the density of the alkylated imidazole groups on the transfection activity mediated by PVIm-R/DNA complexes. As PVIm-R, 7 mol % butylated PVIm (white bars), 23 mol % butylated PVIm (gray bars), or 38 mol % butylated PVIm (black bars) was used for the transfection. The PVIm-Bu/DNA complexes were prepared at the +/- ratio of 4, 8, 12, 24, or 36. Other experimental conditions are the same as those described in Figure 3.
µg/mL. These results suggest that PVIm-R with a relatively short length of alkyl groups such as butyl, ethyl, or methyl groups promises to be a noncytotoxic gene carrier. The noncytotoxic property may be attributed to the partial shielding of surface charge, as poly(ethylene glycol) (29), by unmodified imidazole groups. However, PVIm-R with a relatively long length of alkyl groups such as octyl groups is considered to cause significant cytotoxicity by the presence of the more amphiphilic property for cell membrane damage. Actually, a little significant cytotoxicity was observed when PVIm-Bu was added up to the concentration of 1000 µg/mL, where PVIm-Me and PVIm-Et exhibited no significant cytotoxicity (Figure S-2, Supporting Information). Gene Delivery by PVIm-R/DNA Complexes. As a result of no apparent cytotoxicity, we further examined the gene expression mediated by PVIm-R/DNA complexes in view of the density of alkylated imidazole groups. As shown in Figure 6, the PVIm-Bu polycations with different densities of butylated imidazole groups were used as representative PVIm-R for DNA complex formation. Little gene expression was observed at any +/- ratio when we used the DNA complexes with PVIm-Bu with a lower density (7 mol %) of butylated imidazole groups. In case of the PVIm-Bu with a higher density (38 mol %) of butylated imidazole groups, however, the DNA complexes mediated remarkable gene expression even if the complexes at the +/- ratio of 4 was used. Although the DNA complexes with PVIm-Bu with a middle density (23 mol %) of butylated imidazole groups mediated little gene expression at the +/ratio of 4, the DNA complexes at higher +/- ratios succeeded
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Figure 7. Effect of the length of the alkylated imidazole groups on the transfection activity mediated by PVIm-R/DNA complexes. As PVImR, 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/DNA complexes were prepared at the +/- ratio of 4, 12, or 36. Other experimental conditions are the same those described in Figure 3.
in remarkable gene expression. These results suggest that the density of the butylated imidazole groups in PVIm-Bu was an important factor for gene delivery. As a result of the dependency of the density of the butylated imidazole groups, we finally examined the gene expression mediated by the PVIm-R/DNA complexes in view of the alkyl chain length of alkylated imidazole groups. Figure 7 shows the effect 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 because of too stable retention of DNA (Figure 4) and significant cytotoxicity (Figure 5). As a result of PVIm-Bu with a middle density (18 mol %, Figure 3; 23 mol %, Figure 6) of alkylated imidazole groups, the gene expression mediated by the PVImBu/DNA complexes depended on the +/- ratio. It should be noted that PVIm-Me and PVIm-Et mediated higher gene expression than PVIm-Bu at lower +/- ratios. Especially, the gene expression mediated by the PVIm-Et/DNA complexes did not depend on the +/- ratio. These results suggest that the alkyl chain length of the alkylated imidazole groups was also an important factor for gene delivery. Gene expression decreased with a decrease in the density of the alkylated (butylated) imidazole groups (Figure 6). The higher gene expression mediated by PVIm-Me and PVIm-Et is therefore unexpected (Figure 7) because the shorter length of the alkyl chain decreased the apparent density of the alkylated imidazole groups. However, it is surprising that the PVIm-Bu/ DNA complex with a middle density of butylated imidazole groups was more stable than that with higher density (Figure S-3, Supporting Information). The excess butylated imidazole groups are therefore considered to enhance the membrane disruptive activity to escape from acidic endosomal vesicles. Furthermore, it is also surprising that the DNA complex with PVIm-Me or PVIm-Et was more stable than that with PVImBu (Figure S-4, Supporting Information). At present, the stability mechanism is unclear; analysis is now in progress for more detailed investigations. Nevertheless, it can be said that the PVIm-Et/DNA complex as well as the PVIm-Me complex has more adequate retention of DNA for gene delivery, as compared with the PVIm-Bu/DNA complex. Although amphiphilic imidazolinium compounds for gene delivery were previously reported, the compounds form liposomes whose stability depends on the length of the dialkyl groups of imidazolinium (30). In this study, many alkyl groups in PVIm-R are considered to be intertwined with DNA grooves and the degree of intertwining may affect the transcription of the delivered gene.
Alkylated Poly(1-vinylimidazole)
Taking these results into account, alkylated imidazole groups are essential and unique functional groups in PVIm-R for efficient gene delivery. Conclusions. We have synthesized a unique pH-sensitive polycation PVIm-R and evaluated the physicochemical and biochemical properties of the pH-sensitive gene carrier. The resulting PVIm-R was water-soluble in spite of the deprotonation of the imidazole groups at physiological pH. PVIm-R/DNA complex formation was mediated by alkylated imidazole groups working as anchor groups to retain DNA. The DNA complex with butylated PVIm (PVIm-Bu) formed the domain with a hydrophobic nature, which increased after the protonation of imidazole groups of PVIm-Bu. The increased hydrophobichydrophilic incline of the PVIm-R/DNA complexes at endosomal pH enhanced membrane disruptive activity. PVIm-Bu exhibited no significant cytotoxicity in spite of the existence of cationic groups and easily released DNA, as compared with PVIm-Oc. By using PVIm-R as a pH-sensitive DNA carrier, PVIm-R/DNA complexes mediated efficient gene delivery attributed to the alkylated imidazole groups, and gene expression depended on the length and density of the alkyl chains. Consequently, PVIm-R’s control of the stability of DNA polyion complexes enhanced noncytotoxic gene delivery by optimized alkylated imidazole groups. The control of the polycation/DNA complex properties by varying the length and density of the alkyl chains grafted onto PVIm is expected to offer unique designs for gene delivery systems. Supporting Information Available: Analysis of the formation of the complex between DNA and PVIm-Bu by agarose gel electrophoresis in the absence of EtBr; effect of PVIm-R on the viability of HepG2 cells after 1 day incubation; release of DNA from PVIm-Bu/DNA complexes by dextran sulfates (DS) as assessed by agarose gel electrophoresis; and release of DNA from PVIm-R/DNA complexes by dextran sulfates (DS) as assessed by agarose gel electrophoresis. This material is available free of charge via the Internet at http:// pubs.acs.org.
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