Synthesis and Characterization of Methylated Poly (l-histidine) To

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Synthesis and Characterization of Methylated Poly(L-histidine) To Control the Stability of Its siRNA Polyion Complexes for RNAi Shoichiro Asayama,* Takao Kumagai, and Hiroyoshi Kawakami Department of Applied Chemistry, Tokyo Metropolitan University, 1-1 Minami-Osawa, Hachioji, Tokyo 192-0397, Japan S Supporting Information *

ABSTRACT: Poly(L-histidine) (PLH) with dimethylimidazole groups has been synthesized as a pH-sensitive polypeptide to control the stability of its small interfering RNA (siRNA) polyion complexes for RNA interference (RNAi). The resulting methylated PLH (PLH-Me) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid−base titration and solution turbidity measurement. Agarose gel retardation assay proved that the quaternary dimethylimidazole groups worked as cationic groups to retain siRNA. The stability of the PLH-Me/siRNA complexes has depended on the content of hydrophobic groups, that is, τ/π-methylimidazole groups as well as deprotonated imidazole groups. PLH-Me exhibited no significant cytotoxicity despite the existence of cationic dimethylimidazole groups. By use of PLH-Me as a pH-sensitive siRNA carrier, the PLH-Me/siRNA complexes mediated efficient siRNA delivery attributed to the dimethylimidazole groups, and the gene silencing depended on the content balance among dimethyl, τ/π-methyl, and unmodified imidazole groups. These results suggest that PLH-Me controls the stability of siRNA polyion complexes by enhancing noncytotoxic siRNA delivery by optimizing the content balance of dimethyl, τ/π-methyl, and unmodified imidazole groups.

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INTRODUCTION Small interfering RNA (siRNA) has drawn much attention because of its specific and effective gene silencing action on the expression of a specific target protein in a posttranscriptional mRNA level.1,2 The siRNA composed of a double-strand RNA with 21 nucleotide base pairs specifically degrades a target mRNA with the help of a RNA induced silencing complex (RISC), resulting in the inhibition of the synthesis of the target protein. In siRNA delivery systems, the formation of polycation/siRNA polyion complexes is a key factor for new design of efficient delivery.3−5 Because siRNA has a short structure compared to plasmid DNA, the polyion complexes between siRNA and oppositely charged polycations lack stability under physiological conditions. Therefore, stabilization of the polycation/siRNA polyion complexes is essential for successful siRNA delivery, whereas the polyion complexes are required to release siRNA for its gene silencing effect in the cytoplasm after endosomal escape. For fulfillment of the above inconsistent properties, many efforts to design and chemical modification of polycations have been reported.6−13 © 2012 American Chemical Society

Furthermore, the polycations modified with the histidine or other moieties containing an imidazole group have the ability to escape from an endosome by a proton sponge mechanism.14−19 The imidazole heterocycles displaying a pKa of ∼6 possess a buffering capacity in endosomal pH, inducing membrane destabilization after their protonation. The resulting imidazole groups are therefore considered to facilitate the release of the polycation/siRNA complexes to cytosol. We have already reported carboxymethyl poly(L-histidine) as a pH-sensitive polypeptide to enhance polyplex gene delivery.20 The resulting carboxymethyl poly(L-histidine) possesses dicarboxymethyl, τ/π-carboxymethyl, and unmodified imidazole groups. Moreover, we have already reported alkylated poly(1-vinylimidazole) to control of the stability of the polyion complexes by the length and density of the alkylated imidazole groups, where the methylimidazole groups form the most stable polyion complexes with the highest gene expression.21 Received: January 31, 2012 Revised: June 9, 2012 Published: June 10, 2012 1437

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using a Spectra/Por 7 membrane (molecular weight cutoff of 103). The resulting polypeptide (3) was obtained by freezedrying. Gel Filtration Chromatography (GFC). GFC was carried out using a JASCO PU-980 pumping system (Tokyo, Japan) at a 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 0.3 M Na2SO4 was used as a mobile phase. An amount of 100 μL of 1 mg/mL sample was 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 polypeptide (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). Amino Acid Analysis. Each polypeptide (1 mg) was dissolved in 500 μL of 6 M HCl and hydrolyzed at 110 °C for 24 h. The hydrolysate was diluted to 3 mL with water. The 100 μL of the diluted hydrolysate was then dried, followed by the addition of 800 μL of 0.2 M sodium citrate buffer (pH 2.2). After filtration (0.45 μm), the resulting samples were injected into an ion-exchange column (Hitach High-Technologies Co., Tokyo, Japan) with sodium citrate buffer (pH 3.2−4.9) as a mobile phase. The elution was gradually carried out with changing pH at a flow rate of 0.4 mL/min and was analyzed by the ninhydrin method. Acid−Base Titration and Turbidity Measurement of PLH-Me. To 1.5 mL of an aqueous solution of the polypeptide (3 mg/mL) was added a 0.5 M HCl solution, and the acidic polypeptide solution (pH 2) was titrated with a 0.5 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 0.5 M NaOH and stopped at pH 12. The turbidity of the solution during the titration was measured by monitoring the absorbance at 500 nm with a spectrophotometer (model V-660, JASCO, Tokyo, Japan). Agarose Gel Retardation Assay. The PLH-Me and siRNA were mixed in 50 mM sodium phosphate buffer (pH 7.4) at various polypeptide/DNA ratios (positive/negative = 0−32) as follows: The siRNA stock solution (1.93 μL) was diluted with the phosphate buffer (pH 7.4). To the resulting siRNA solution was added a stock solution of the polypeptide (1−4 μL) at various polypeptide/siRNA ratios. The final volume of the mixture was adjusted to 13.5 μL. After 5 min of incubation at room temperature, 13.5 μL of each sample (corresponding to 500 ng of siRNA) was mixed with a loading buffer (1.5 μL) and loaded onto a 1% agarose gel containing 1 μg/mL ethidium bromide (EtBr). Gel electrophoresis was run at room temperature in 50 mM sodium phosphate buffer (pH 7.4) at 50 V for 15 min. The siRNA bands were visualized under UV irradiation. In the case of assay for the stability of the PLH-Me/siRNA complexes, gel electrophoresis was run in the presence of dextran sulfate (1−6 mM as sulfate group) incubated with each sample at room temperature for 10 min. 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 fetal bovine serum (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 48 h at 37 °C. By further

The above background has led us to synthesize the poly(Lhistidine) with dimethylimidazole, τ/π-methyl, and unmodified imidazole groups, that is, methylated poly(L-histidine), as a new pH-sensitive polypeptide to control the stability of its siRNA polyion complexes for efficient RNA interference (RNAi). The control of the siRNA polyion complex stability for efficient RNAi by the content balance among dimethyl, τ/π-methyl, and unmodified imidazole groups has no precedent, to the best of our knowledge. Consequently, the pH-sensitive methylated poly(L-histidine), that is, PLH-Me, to control the stability of siRNA polyion complexes is expected to mediate efficient gene silencing by optimizing the content balance among dimethyl, τ/ π-methyl, and unmodified imidazole groups in the polypeptide backbone.

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EXPERIMENTAL PROCEDURES Materials. Poly(L-histidine) (PLH) hydrochloride (Mw ≥ 5000), poly(L-lysine) (PLL) hydrobromide (Mw, 4000− 15000), and poly(ethylenimine) (PEI) solution (Mw ≈ 750000) were purchased from Sigma-Aldrich Co. LLC. Iodomethane was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Synthetic siRNA for luciferase GL3 was obtained from Gene Design Inc. (Osaka, Japan), and the sequence was CUUACGCUGAGUACUUCGA (19-mer) for sense and UCGAAGUACUCAGCGUAAG (19-mer) for antisense with dTdT-overhang at two terminal ends. The scrambled siRNA for a negative control was also from Gene Design Inc. and the sequence was UUCUCAGAACGUGUCACGU (19-mer) for sense and ACGUGACACGUUCUGAGAA (19-mer) for antisense with dTdT-overhang. All other chemicals of a special grade were used without further purification. Synthesis of PLH-Me. A typical procedure is as follows (Scheme 1): PLH hydrochloride (1) (50 mg) was dissolved in Scheme 1. Synthesis of Methylated Poly(L-histidine)

4 mL of water, which was brought to pH 5 by the addition of 1 M NaOH. To the resulting PLH solution was added the iodomethane (2) (100 μL) dissolved in 2 mL of N,Ndimethylformamide (DMF). The reaction mixture was incubated at 40 °C for 24 h. After the incubation, the iodomethane (2) (100 μL) dissolved in 2 mL of DMF was added to the reaction mixture again. For further modification, the iodomethane (2) (100 μL) in 2 mL of DMF was repeatedly (four times or six times) added to the reaction mixture, around pH 6. The resulting reaction mixture was further incubated at 40 °C for 24 h, followed by the dialysis against distilled water 1438

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incubation for 4 h, the cell viability was measured using the Alamar Blue assay22 in triplicate. RNA Interference. 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% heatinactivated 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 PEI. After 1 day of incubation, the medium containing PEI/pGL3 complexes was removed and the cells were thoroughly washed twice with the medium supplemented with 10% FBS. PLH-Me/siRNA complexes were prepared by mixing various amounts (1−10 μL) of PLH-Me stock solutions in PBS (−) with anti-luciferase GL3 siRNA or nonspecific scrambled control siRNA solutions (195 ng in 5.25 μL) to obtain various positive/negative ratios in a final volume of 105 μL (per 7 wells), followed by 5 min of incubation at room temperature. Then an amount of 15 μL (per 1 well) of the resulting PLH-Me/siRNA complexes was added to the above washed cells to obtain a final siRNA concentration of 100 nM. After 2 days of incubation without washing, the cells were subjected to the luciferase assay (Promega kit) according to the manufacturer's instruction. Luciferase activities were normalized by protein concentrations and are presented as relative light unit (RLU). Protein concentrations were determined by BCA protein assay kit (Pierce) according to the manufacturer's instruction.

Figure 1. (A) Acid−base titration curves of PLH-Me: (●) PLHMe(0); (□) PLH-Me(25); (○) PLH-Me(68); (△) PLH-Me(87). Acidic polypeptide solutions (3 mg/mL) were titrated with the stepwise addition of 0.5 M NaOH. (B) Effect of pH on the solubility of the PLH-Me in water (3 mg/mL): (●) PLH-Me(0); (□) PLHMe(25); (○) PLH-Me(68); (△) PLH-Me(87). The turbidity was measured by monitoring the absorbance at 500 nm of the polypeptide aqueous solution during the acid−base titration.

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RESULTS AND DISCUSSION Synthesis of PLH-Me. To synthesize methylated poly(Lhistidine), as shown in Scheme 1, poly(L-histidine) hydrochloride (1) was reacted with iodomethane (2) to obtain quaternary imidazole groups, that is, cationic dimethylimidazole groups. The GFC profile of the resulting polypeptides (3) indicated that the number-average molecular weight (Mn) of the resulting polypeptide (3) was about 1.2 × 104. The 1H NMR spectrum of the resulting polypeptides (3) showed the characteristic signals of both poly(L-histidine) backbone [βmethylene (δ 2.6−3.0 ppm), α-methine (δ 4.3−4.6 ppm), and imidazole (δ 6.6−7.1, 7.3−7.8, and 8.4−8.5 ppm)] and methyl groups (δ 3.3−3.7 ppm) (Figure S-1A, Supporting Information). However, the amount of dimethyl histidine, τ-methyl isomer, and π-methyl isomer, respectively, is unclear, so that amino acid analysis was carried out. From the chromatograms, the content of dimethyl histidine is estimated to be 25 mol % (τ-methyl, 16 mol %; π-methyl, 17 mol %), 68 mol % (τmethyl, 16 mol %; π-methyl, 8 mol %), and 87 mol % (τmethyl, 7 mol %; π-methyl, 4 mol %) (Figure S-1B, Supporting Information). Thus, we have synthesized various poly(Lhistidine) biomacromolecules with cationic dimethylimidazole groups, that is, “PLH-Me.” Namely, we call the PLH with 25, 68, and 87 mol % dimethyl hitidine PLH-Me(25), PLHMe(68), and PLH-Me(87), respectively. pH-Dependent Behavior of PLH-Me in Water. To examine the ionic properties of the remaining imidazole groups, we carried out the acid−base titration of the resulting PLH-Me solution, as shown in Figure 1A. The imidazole protons of the PLH-Me were gently dissociated around pH 6 so that the pKa of the PLH-Me is considered to be around 6. The proton dissociation profile approximately depended on the content of dimethylimidazole groups. Namely, the PLH-Me(25) exhibited larger capacity of proton buffering around pH 6 because the PLH-Me(25) had more unmodified imidazole groups with a

pKa of ∼6. On the other hand, almost no proton buffering of the PLH-Me(87) was observed around pH 6, owing to almost complete modification of imidazole groups to form quaternary dimethylimidazole groups. From these titration results, the following positive/negative charge ratio of PLH-Me/siRNA complexes will be estimated as a ratio of the quaternary dimethylimidazole groups of PLH-Me to the phosphate groups of siRNA at pH 7.4. The solution behavior of the PLH-Me, as shown in Figure 1B, is totally different from that of the PLH, that is, PLHMe(0). The PLH-Me(0) exhibits precipitation above pH 6.0 owing to the deprotonation of the imidazole groups of the PLH-Me(0); namely, the PLH-Me(0) is not water-soluble. Therefore, the PLH-Me(0) is not available as siRNA carrier. On the other hand, the aqueous solution of the PLH-Me(25) exhibited no significant turbidity above pH 6 in spite of the deprotonation of unmodified imidazole groups (Figure 1A). The introduction of the cationic dimethylimidazole groups, therefore, improves the water solubility of the PLH at physiological pH. Complex Formation between PLH-Me and siRNA. We examined whether the PLH-Me formed the polyion complexes with siRNA by agarose gel electrophoresis (Figure 2). The complete retardation of siRNA was observed at a positive/ negative charge ratio of 32. However, under experimental conditions, significant free siRNA was observed in spite of the mixing at a positive/negative charge ratio of 2. Under same experimental conditions, instead of siRNA, no free DNA was observed at a positive/negative charge ratio of 1 (results not 1439

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Me(25)/siRNA complexes, few siRNA migrated even in the presence of the higher concentration of the dextran sulfate (lane 8). These results suggest that the PLH-Me(87)/siRNA complexes easily released the siRNA by exposure to polyanions and that the PLH-Me(25)/siRNA complexes stably retained the siRNA. Moreover, when we determined the particle size and ζ potential of PLH-Me/siRNA complexes (Table S-1, Supporting Information), a significant value was obtained in the only case of the PLH-Me(25)/siRNA complexes. This is probably caused by adequate content of methyl (τ-methyl and π-methyl) imidazole groups as hydrophobic moiety to stabilize the electrostatic interaction between siRNA and dimethylimidazole groups of the PLH-Me. In this study, the stability of polycation/siRNA complexes has depended on the content of hydrophobic groups, that is, methylimidazole groups as well as deprotonated imidazole groups, in the polycation, promising unique design of polycation/siRNA complexes. We have therefore considered that the PLH-Me(25)/siRNA complexes have the ability to retain the siRNA stably outside the target cell for RNAi. Cytotoxicity of PLH-Me/siRNA Complexes. Cytotoxicity of a siRNA carrier is an important factor for clinical applications. After the endosomal escape of siRNA mediated by the free polycations that has been internalized at the same time as the siRNA polyion complexes, in general, free polycations also exist solely during the release of siRNA from the polyion complexes. 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 estimation of the interaction of the polycations with cells rather than that of the polyion complexes with siRNA. As shown in Figure 4, we

Figure 2. Analysis of the formation of the complexes between PLHMe and siRNA by agarose gel electrophoresis, showing the interaction of each PLH-Me (carrier) with siRNA at pH 7.4: lane 1, siRNA alone; lanes 2−6, PLH-Me/siRNA mixtures at different charge ratios relative to dimethylimidazole groups of PLH-Me per phosphate group of siRNA (+/− = 2 (lane 2), 4 (lane 3), 8 (lane 4), 16 (lane 5), or 32 (lane 6)).

shown). Therefore, these results are consistent with less formation of the PLH-Me/siRNA polyion complexes because of the lower molecular weight of siRNA, compared with DNA. Stability of PLH-Me/siRNA Complexes. To examine further the stability of the PLH-Me/siRNA complexes, we attempted to release siRNA from the polyion complexes by competitive exchange with other polyanions.23 For effective RNA interference (RNAi), the release of siRNA should not happen outside the target cell, whereas that must occur somewhere inside to allow binding of the RNAi 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 PLH-Me/siRNA complexes at a positive/negative charge ratio of 32 were incubated with dextran sulfates. The results are shown in Figure 3. As the concentration of the dextran sulfate increased, the siRNA increasingly migrated (lanes 6−8) in the case of the PLHMe(87)/siRNA complexes. Furthermore, the siRNA complexes was hard to migrate in the case of the PLH-Me(68)/siRNA complexes, as compared with the PLH-Me(87)/siRNA complexes. On the other hand, in the case of the PLH-

Figure 4. Effect of PLH-Me on the viability of HepG2 cells after 48 h incubation: (□) PLH-Me(25); (○) PLH-Me(68); (△) PLH-Me(87); (■) PLL; (●) PEI. Symbols and error bars represent the mean and standard deviation of the measurements made in triplicate wells.

therefore examined the effect of the PLH-Me on the cell viability. The viability of HepG2 hepatoma cells did not significantly decrease when all PLH-Me was added up to the concentration of 400 μg/mL, which was higher than the RNAi conditions. Consequently, it is worth noting that the PLH-Me exhibited no apparent cytotoxicity in spite of the content of the cationic dimethylimidazole groups. On the other hand, little viability was observed when the control PEI with higher molecular weight (Mn ≈ 60000) was added up to 50 μg/mL. The control cationic polypeptide PLL (Mn = 1.0 × 104) with the almost same molecular weight of the PLH-Me exhibited significant cytotoxicity at 200 μg/mL. In view of a positive charge concentration, the cytotoxic concentration of both PEI

Figure 3. Release of siRNA from PLH-Me/siRNA complexes by dextran sulfates (DS) as assessed by agarose gel electrophoresis: lane 1, siRNA alone; lane 2, 0 mM; lane 3, 1 mM; lane 4, 2 mM; lane 5, 3 mM; lane 6, 4 mM; lane 7, 5 mM; lane 8, 6 mM. The siRNA mixtures with each PLH-Me (carrier) at a positive/negative charge ratio of 32 were incubated for 10 min at room temperature in the presence (lanes 3−8) or absence (lane 2) of DS (1−6 mM as sulfate group), followed by loading to the gel. 1440

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that the PLH-Me(25) enhanced the cellular uptake and endosomal escape of siRNA for efficient RNAi (Figure S-4, Supporting Information). Taking these results into account, the dimethylimidazole groups are essential and unique functional groups in the PLH-Me for efficient siRNA delivery. Furthermore, the content balance among dimethyl, τ/π-methyl, and unmodified imidazole groups is considered to be important for efficient gene silencing by siRNA.

and PLL was also lower than that of PLH-Me (Figure S-2, Supporting Information). These results suggest that the PLHMe polypeptide with dimethylimidazole groups promises to be a noncytotoxic siRNA carrier; namely, dimethylimidazole groups are considered to be noncytotoxic cationic species. RNAi by PLH-Me/siRNA Complexes. As a result of no apparent cytotoxicity, we further examined the RNAi mediated by the PLH-Me/siRNA complexes in view of the content of dimethylimidazole groups. As shown in Figure 5 (also see

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CONCLUSION We have synthesized the unique pH-sensitive polypeptide PLH-Me and evaluated the physicochemical and biochemical properties of the siRNA carrier to control the stability of its siRNA polyion complexes for RNAi. The resulting PLH-Me was water-soluble in spite of the deprotonation of the imidazole groups at physiological pH. The PLH-Me/siRNA complex formation was mediated by the quaternary dimethylimidazole groups working as cationic groups to retain siRNA. The stability of the PLH-Me/siRNA complexes has depended on the content of hydrophobic groups, that is, τ/π-methylimidazole groups as well as deprotonated imidazole groups. The PLH-Me exhibited no significant cytotoxicity in spite of the existence of cationic dimethylimidazole groups. By use of PLHMe as a pH-sensitive siRNA carrier, the PLH-Me/siRNA complexes mediated efficient siRNA delivery attributed to the dimethylimidazole groups, and the gene silencing depended on the content balance among dimethyl, τ/π-methyl, and unmodified imidazole groups. Consequently, the PLH-Me to control the stability of siRNA polyion complexes enhanced the noncytotoxic siRNA delivery by the optimized content of dimethyl, τ/π-methyl, and unmodified imidazole groups. The control of the polycation/siRNA complexes properties by varying the content balance of the methylation of the imidazole groups of pH-sensitive poly(L-hisitidine) biomacromolecules is expected to offer a unique design for siRNA delivery systems.

Figure 5. Luciferase gene knockdown by PLH-Me/siRNA complexes. The PLH-Me polypeptides with different contents of the dimethyl histidine were formed with siRNA at a positive/negative charge ratio of 8 (gray bars) or 32 (black bars). PEI was used as a positive control. The data were represented as inhibition ratio of siRNA for luciferase GL3 to scrambled siRNA. In particular, the PLH-Me(25)/siRNA complexes at a positive/negative charge ratio of 32 are statistically different, p < 0.005 using t-test. Symbols and error bars represent the mean and standard deviation of the measurements made in triplicate wells.

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Figure S-3, Supporting Information), the PLH-Me polycations with different content of dimethylimidazole groups were used for the siRNA complex formation. No gene silencing efficacy was observed at any positive/negative charge ratio when we used the siRNA complexes with the PLH-Me(87). In the case of the PLH-Me(68), on the other hand, the siRNA complexes mediated little gene silencing at a positive/negative charge ratio of 32. The PLH-Me(25)/siRNA complexes mediated significant gene silencing (65%) at a positive/negative charge ratio of 32 without cytotoxicity (70 μg/mL, see Figure 4). Although the control PEI/siRNA complexes mediated remarkable gene silencing (98%) at a positive/negative charge ratio of 32,24 cytotoxicity was observed under the experimental conditions (11 μg/mL, see Figure 4). Therefore, the resulting RNAi may be partially due to the cytotoxic property of PEI. These results suggest that the content of the dimethylimidazole groups in the PLH-Me was an important factor for RNAi. The PLH-Me(25)/ siRNA complexes exhibited the most stable retention of siRNA in the PLH-Me at a positive/negative charge ratio of 32 (Figure 3), due to the hydrophobic groups (τ/π-methylimidazole groups as well as deprotonated imidazole groups). Furthermore, it can be said that the PLH-Me(25)/siRNA complexes maintain the stable retention of siRNA in the presence of 10% serum (Figure 5). For efficient gene silencing, the endosomal escape of siRNA, caused by the protonation of imidazole groups, is also necessary. Our preliminary study of the intracellular trafficking of the PLH-Me(25)/siRNA complexes by observation with a confocal laser scanning microscope shows

ASSOCIATED CONTENT

* Supporting Information S

Representative 1H NMR spectrum of the PLH-Me; chromatograms (amino acid analysis) of the various PLH-Me polypeptides; particle size and ζ potential of PLH-Me/siRNA complexes; effect of PLH-Me on the viability of HepG2 cells; luciferase gene knockdown (inhibition ratio as the % of the untreated cells) by PLH-Me/siRNA complexes; confocal laser scanning microscope images showing intracellular trafficking of the PLH-Me/siRNA complexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +81-42-677-1111, extension 4976. Fax: +81-42677-2821. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Professor Yukihiko Aramaki, Associate Professor Yoichi Negishi, and Assistant Professor Yoko EndoTakahashi of the Tokyo University of Pharmacy and Life Sciences for use the confocal laser scanning microscope with their helpful assistance. 1441

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dx.doi.org/10.1021/bc300044r | Bioconjugate Chem. 2012, 23, 1437−1442