Efficient Gene Transfection by Histidine-Modified Chitosan through

May 25, 2010 - Institute for the Integrated Cell-Material Sciences. ..... **p < 0.01 (n = 3, +Bafilomycin A1 vs −Bafilomycin A1, Student's t test). ...
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Bioconjugate Chem. 2010, 21, 1087–1095

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Efficient Gene Transfection by Histidine-Modified Chitosan through Enhancement of Endosomal Escape Kai-Ling Chang,† Yuriko Higuchi,†,‡ Shigeru Kawakami,† Fumiyoshi Yamashita,† and Mitsuru Hashida*,†,§ Department of Drug Delivery Research and Institute for Innovative NanoBio Drug Discovery and Development, Graduate School of Pharmaceutical Sciences, and Institute for the Integrated Cell-Material Sciences, Kyoto University, Japan. Received February 2, 2010; Revised Manuscript Received May 1, 2010

Chitosan has the potential to be a biocompatible gene carrier. However, the transfection efficiency of chitosan is low because of the slow endosomal escape rate. The buffering capacity of histidine in the endosomal pH range would help the escape of plasmid DNA (pDNA) from endosomes. In this study, histidine was introduced into chitosan to improve the transfection efficiency. Chitosan and histidine were linked by disulfide bonds provided by 2-iminothiolane and cysteine. The complexes were prepared by mixing chitosan or histidine-modified chitosan with plasmid DNA. A broader buffering range of histidine-modified chitosan was observed, and the cellular uptake of histidine-modified chitosan/pDNA complexes was higher than that of chitosan/pDNA complexes. Although chitosan/tetramethylrhodamine (TMR)-pDNA complexes were trapped in the vesicles in cytosol, TMRpDNA carried by histidine-modified chitosan was more widely distributed in the cytosol. This result suggests that histidine can help pDNA escape from endosomes with the help of the high buffering capacity. The gene expression of histidine-modified chitosan/pDNA complexes was higher than that of chitosan/pDNA complexes. These results suggest that histidine modification improves the transfection efficiency of chitosan.

INTRODUCTION Chitosan, which is a cationic polysaccharide, has played an important role in biomedicine for more than a decade, being used as a food additive (1) and a wound dressing to assist healing (2). It is also well-known that chitosan has the potential to act as a gene carrier (3–6). The amino groups in the structure of chitosan provide positive charges to form complexes with the negative charges of nucleic acids. The outstanding biocompatibility of chitosan is also an advantage for biomedical applications (3). A number of studies have proven that chitosan can be used in vivo with negligible side effects (7, 8). These advantages make chitosan a promising material for use as a gene carrier. However, the gene transfection efficiency of chitosan as a carrier is very low compared with conventional nonviral gene carriers,includingliposomes(4,9,10)andpolyethyleneimines(6,11). One reason for the low gene expression efficiency of chitosan is its slow endosomal escape rate (6, 12). In fact, chitosan effectively protects and introduces nucleic acids into cells. However, endosomes would be the next barrier to the gene delivery of chitosan. Ko¨ping-Ho¨ggård et al. showed that chitosan was trapped in endosomes over 24 h (6). Therefore, the enzymes in endosomes and lysosomes would gradually degrade the nucleic acids in endosomes or lysosomes over a long period. Other problems including low solubility under physiological conditions and complicated physicochemical properties are also major concerns for the efficient gene transfection of chitosan (13). These problems, especially the slow endosomal escape * To whom correspondence should be addressed. Dr. Mitsuru Hashida, Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-shimoadachi, Sakyo-ku, Kyoto 606-8501, Japan. Tel: +075-753-4525, Fax: +075-753-4575, E-mail: [email protected]. † Department of Drug Delivery Research. ‡ Institute for Innovative NanoBio Drug Discovery and Development. § Institute for the Integrated Cell-Material Sciences.

rate, need to be solved to enhance the transfection efficiency of chitosan prior to its application as a gene carrier. There are several strategies to overcome the endosome barrier. For example, chloroquine, an endosomolytic reagent, can accumulate in lysosomes and disrupt them by increasing the pH (14, 15), and pH-sensitive liposomes can escape from endosomes due to membrane disturbance (16, 17). Histidine, a nonessential amino acid, contains an imidazole ring (pKa3 6.04) which increases the buffering capacity in endosomes and lysosomes. Histidine and histidine-containing dipeptides, such as carosine and anserine, are known to act as endogenous buffers in skeletal muscle (18, 19). For this reason, histidine is commonly used as a modifying group to improve the endosomal escaping rate of several nonviral gene carriers, such as polylysine, liposomes, and poly(amino acid) derivatives (20–24). We have demonstrated that histidine can enhance the gene transfection efficiency of liposomes by increasing the buffering capacity (21). In addition, the conjugation site on the chitosan structure and the hydrophilicity of histidine itself may help the dissolution of chitosan. The purpose of this study was to increase the gene transfection efficiency of chitosan/pDNA complexes by enhancing the endosomal escape rate. In this study, we introduced histidine to the amino groups of chitosan via disulfide bonds. The degree of substitution of histidine and the buffering capacity of histidine-modified chitosan were measured. The physicochemical properties of the complexes were evaluated by determination of the particle size and zeta potential, as well as agarose gel electrophoresis. The gene transfection process was examined by confocal microscopy, flow cytometry, and luciferase assay in order to prove the enhanced gene transfection of histidinemodified chitosan by increased cellular uptake and a buffering effect.

EXPERIMENTAL PROCEDURES Materials. Highly purified chitosan (number-average molecular weight: 173 kDa, degree of deacetylation: 88%) was

10.1021/bc1000609  2010 American Chemical Society Published on Web 05/25/2010

1088 Bioconjugate Chem., Vol. 21, No. 6, 2010 Scheme 1. Histidine-Cysteine-OMe (His-Cys-OMe) Synthesis

purchased from SEIKAGAKU Biobusiness (Tokyo, Japan). Cys(Acm)-OH · HCl, Boc-His(Boc)-OH, and hydroxybenzotriazole · H2O (HOBt · H2O) were purchased from Watanabe Chemical Industries (Hiroshima, Japan). O-BenzotriazoleN,N,N′,N′-tetramethyl-uronium-hexafluorophosphate (HBTU) was purchased from Peptide Institute (Osaka, Japan). Thionyl chloride (SOCl2), deuterium oxide (D2O), sodium bicarbonate (NaHCO3), citric acid monohydrate, sodium chloride, chloroform, methanol, ethanol, diethyl ether, trifluoroacetic acid (TFA), potassium diphosphate (KH2PO4), dimethyl sulfoxide (DMSO), sodium acetate · 3H2O, ninhydrin, hydrindantin · 2H2O, and sodium hydroxide (NaOH) were purchased from Nacalai Tesque (Kytoto, Japan). Dehydrated dimethylformamide (DMF), petroleum ether, L-histidine, glucose, magnesium chloride (MgCl2), and 4% paraformaldehyde were purchased from Wako Pure Chemical Industries (Osaka, Japan). Mercury(II) acetate was purchased from Sigma-Aldrich Corporation (Saint Louis, MO). 2-iminothiolane · HCl was purchased from Pierce (Rockford, IL). Alexa Fluor 488 transferrin conjugate, LysoSensor Green DND-189, LysoTracker Green DND-26, Lipofectamine 2000, Opti-MEM I reduced serum medium, Trypan blue stain prepared in 0.85% NaCl, and SYBR Gold nucleic acid gel stain were purchased from Invitrogen (Carlsbad, CA). Agarose X and S were purchased from NIPPON GENE (Osaka, Japan). Label IT fluorescein nucleic acid labeling kit and Label IT TMrhodamine nucleic acid labeling kit were purchased from Mirus Bio (Madison, WI). jetPEI was purchased from PolyplusTransfection Inc. (New York, NY). PicaGene luciferase assay Scheme 2. Chitosan-Cysteine-Histidine (Chi-CH) synthesis

Chang et al.

kit was purchased from Toyo Corporation (Tokyo, Japan). All solvent and reagents were ACS grade and used directly without further treatment. His-Cys-OMe Synthesis (Scheme 1). Cys(Acm)-OH · HCl was reacted with SOCl2 to form the methyl ester. Twenty milliliters of methanol was cooled to -30 °C, and then SOCl2 was added to the methanol dropwise. After cooling on ice, the mixture was added with 1 g Cys(Acm)-OH and then stirred at room temperature for 15 h. The methanol in the mixture was removed in an evaporator, and the product, Cys(Acm)-OMe, was recrystallized from ethanol and cold diethyl ether. The coupling reaction of Cys(Acm)-OMe and Boc-His(Boc)-OH was conducted with HBTU and HOBt · H2O in DMF for 12 h at room temperature. DMF was removed in an evaporator, and the residue was dissolved in chloroform and washed twice with 5% sodium bicarbonate, 10% citric acid, and saturated sodium chloride. Chloroform was removed in an evaporator and the Boc and Acm-protected product was recrystallized using a mixture of ethanol, petroleum ether, and diethyl ether. The deprotection of the Boc groups and Acm groups was carried out by 95% TFA and mercury(II) acetate treatment, respectively. The obtained His-Cys-OMe was confirmed by ESI-MS (Mariner ESI TOF Biospectrometry Workstation, PerSeptive Biosystems, Inc., Framingham, MA). Synthesis of Histidine-Modified Chitosan (Chi-CH, Scheme 2). The conjugation of chitosan and His-Cys-OMe was carried out according to Masuko et al. (25) with slight modification. Chitosan was dissolved in 1% acetic acid (pH 2.0) and potassium phosphate buffer (50 mM KH2PO4, 2 mM EDTA · 2Na, and 150 mM NaCl, pH 8.0) was mixed with this chitosan solution in the volume ratio of 1:1. Traut’s reagent (2-iminothiolane · HCl) was added to the solution directly, and the reaction mixture was stirred for 4 h at room temperature. After purification by dialysis (1000 molecular weight cutoff) for 48 h, the reaction product was immediately reacted with His-Cys-OMe in different weight ratios to 2-iminothiolane · HCl (Table 1). DMSO was added to the reaction mixture in a volume ratio of 10:1 (reaction mixture/DMSO). The reaction mixture was stirred for 12 h, and then purified by dialysis (1000

Histidine-Modified Chitosan

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Table 1. Reaction Conditions for Chi-CH Synthesis and the Degree of Substitution of His-Cys-OMe in Chitosan reactant 2-iminothiolane · HCl

chitosan

His-Cys-OMe

product

material

(mg)

(mmol)

(mg)

(mmol)

(mg)

(mmol)

degree of substition (%)

Chi-CH A Chi-CH B Chi-CH C

100 100 100

0.0006 0.0006 0.0006

15 35 100

0.1090 0.2543 0.7266

200 200 400

0.7353 0.7353 1.4706

2.72 3.41 4.32

molecular weight cutoff) for 48 h. The reaction mixture after dialysis was freeze-dried. Histidine Quantification. The amount of His-Cys-OMe conjugated to chitosan was measured by the ninhydrin test established by S-W Sun et al. (26). In brief, Chi-CH was dissolved in sodium acetate buffer (100 mM sodium acetate and 100 mM sodium chloride solution, pH 4.5), and the disulfide bonds were reduced using 75 mM dithiothreitol. The reduction reaction was carried out at room temperature for 1 h, and then the chitosan was removed by Microcon centrifugal filters (3000 molecular weight cutoff). The dipeptide solution in Chi-CH was analyzed by the ninhydrin test. Ninhydrin solution was prepared using 20 mg ninhydrin and 33.35 mg hydrindantin · 2H2O dissolved in DMSO/4 N sodium acetate buffer (7.5 mL/2.5 mL) under nitrogen gas. Histidine standard solution (10-100 µg/mL) was prepared in distilled water. The histidine standard solution and the dipeptide solution were mixed with ninhydrin solution in a volume ratio of 1:1, and then heated at 100 °C for 10 min. After heating, the histidine standard solution and the dipeptide solution were placed on ice for 10 min. Fifty milliliters of the dipeptide solution was added to each well of a 96-well plate, followed by 125 µL 50% ethanol. The absorbance of the solution was determined using a microplate reader (BioRad 550 microplate reader, Foster City, CA) at 570 nm. The degree of substitution was calculated based on a histidine standard curve. Measurement of Buffering Capacity. Four micromolar chitosan and Chi-CH solutions were prepared in sodium acetate buffer (pH 5.5). The chitosan and Chi-CH solutions were titrated with 0.1 N NaOH. The pH was recorded using a pH meter (Twin pH AS-212, As one, Osaka, Japan), and the buffering capacity was compared. Preparation and Purification of pCMV-Luc. pCMV-Luc was constructed by subcloning the HindIII/XbaI firefly luciferase cDNA fragment from a pGL3-control vector (Promega, Madison, WI) into the polylinker of a pcDNA3 vector (Invitrogen, Carlsbad, CA). pDNA was amplified in the Escherichia coli strain DH5R, isolated, and purified using a JETSTAR Plasmid Purification Giga Kit (GENOMED GmbH, Lo¨hne, Germany). The concentration of pDNA was measured using a Shimadzu UV-vis spectrometer (Shimadzu, Kyoto, Japan) at 260 nm. Measurement of Particle Size and Zeta Potential. Chitosan or Chi-CH was dissolved into sodium acetate buffer (pH 5.5) at a concentration of 5 mg/mL, and pDNA was dissolved in 5% glucose at a concentration of 1 mg/mL for use as stock solutions. Chitosan or Chi-CH and pDNA solutions were mixed in the same volume ratio with different concentrations based on the A/P ratio (A: amino groups in chitosan structure; P: phosphate groups in pDNA structure). The size and zeta potential of the complexes were measured using a ZetaNanoSizer (Malvern ZetaNanoSizer Nano-ZS, Worcestershire, UK). Evaluation of Complex Stability. The stability of the complexes was evaluated by agarose gel electrophoresis. One microgram of pDNA was mixed with chitosan or Chi-CH at different A/P ratios. The complexes were loaded onto 1% Agarose S gel. For the enzymatic stability assay, the complexes

were incubated with 10 U DNase I (Roche, Manheim, Germany) in 5 mM MgCl2 solution for 3 h at 37 °C. DNase I was quenched by TE buffer after a 3 h incubation, then DNA was extracted using phenol/chloroform/isoamyl alcohol (Nacalai Tesque, Kyoto, Japan). The extracted DNA was loaded onto 5% Agarose X gel. The pDNA on the gel was stained using a SYBR Gold nucleic acid gel stain. pCMV-Luc Labeling. The pDNA was labeled with fluorescein and tetramethylrhodamine (TMR) using a Label IT fluorescein nucleic acid labeling kit or a Label IT TM-rhodamine nucleic acid labeling kit, respectively. The labeling procedure followed the manufacturer’s instructions. Briefly, pDNA was incubated with labeling reagents at 37 °C for 2 h. Then, pDNA was precipitated with 100% ethanol and washed with 70% ethanol. The concentration of fluorescence-labeled pDNA was measured by UV absorbance at 260 nm. Cell Culture and Cellular Uptake. For the uptake study, human embryonic kidney (HEK293) cells (RIKEN Cell Bank, Tsukuba, Japan) were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM Lglutamine (Invitrogen, Carlsbad, CA). HEK293 cells were seeded onto 12 well plates at a density of 3 × 105 cell/mL. After a 24 h incubation, the medium was changed to Opti-MEM I for complex administration. One microgram per milliliter of fluorescein-labeled pDNA complexed with chitosan, Chi-CH, or Lipofectamine 2000 was added to each well. At 2, 6, and 12 h after incubation, Opti-MEM I containing complexes was removed, and the cells were collected in phosphate buffer and centrifuged at 5000 rpm for 10 min. The cell pellets were fixed and resuspended in 70% ethanol and incubated with Trypan blue for the quenching of the extracellular fluorescein. The resuspended cells were repeatedly centrifuged at 5000 rpm for 10 min and resuspended in phosphate buffer for flow cytometry analysis (Becton Dickinson FACSCalibur, Franklin Lakes, NJ). Intracellular Distribution of pDNA. HEK293 cells were used as the cell model to monitor the endosomal escape of the complexes. HEK293 cells were seeded on 12 well plates at a cell density of 2 × 105 cells/mL. After a 24 h incubation, HEK293 cells were incubated with 100 µg/mL Alexa Fluor 488 transferrin conjugate for 20 min in Opti-MEM I medium before addition of the complexes. Lysosomes were labeled with 1 µM LysoSensor Green DND-189 and 1 µM LysoTracker Green DND-26 for 30 min in Opti-MEM I medium before addition of the complexes. The complexes containing 2 µg/mL TMRlabeled pDNA were then added to the cells. After a 6 h incubation, HEK293 cells were washed with phosphate buffer and fixed with 4% paraformaldehyde. The endosomal escape was observed using a Nikon Eclipse Ti inverted confocal microscope equipped with a Nikon EZ-C1 observation system (Nikon, Tokyo, Japan). The images were processed using ImageJ software. The objective was a 60× water immersion objective. Proton Pump Inhibition. HEK293 cells were seeded in 12 well plates at a density of 1.5 × 105 cells/mL and incubated at 37 °C, 5% CO2 for 24 h. Before addition of the complexes, the cells were incubated with 100 nM bafilomycin

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complexes containing 6 µg/mL pDNA in Opti-MEM I for 6 h. The medium was changed to DMEM after a 6 h incubation. The cells were harvested at 72 h and then lysed for the measurement of luciferase activity as described previously. The relative luminescence unit (RLU) was measured by luminometer (Lumat Single Tube Luminometers LB 9507, Berthold Technologies GmbH & Co. KG, Wildbad, Germany) and normalized by the protein content, which was measured by Bradford assay.

RESULTS

Figure 1. Buffering capacity of chitosan and Chi-CH. Chitosan and Chi-CH were determined after dissolution in sodium acetate buffer at pH 5.5 and then titration with 0.1 N NaOH. The pH was plotted to compare the differences in buffering capacity after treatment with different molar ratios of reactants.

A1 in Opti-MEM I for 10 min. Then, 6 µg/mL pDNA complexed with chitosan or Chi-CH was added to each well. Lipofectamine 2000 containing 2 µg/mL pDNA was prepared according to the protocol provided by the manufacturer. The dose of pDNA of jetPEI was reduced to half the dose suggested by the protocol, 1 µg/mL, because of concerns about the toxicity of bafilomycin A1 and polyethylenimine (PEI). The medium was changed to DMEM after a 6 h incubation. The cells were harvested after a 24 h incubation and then lysed. The amount of luciferase was measured using a PicaGene Luciferase Assay Kit. Measurement of Gene Transfection Efficiency. HEK293 cells were seeded in 12-well plates at a density of 1 × 105 cells/mL. After a 24 h incubation, cells were incubated with

Synthesis of Chi-CH. Liquid-phase synthesis was used in this study because a greater amount of dipeptide can be produced by liquid-phase synthesis than solid-phase synthesis. Mass calculated for C10H16N4O3S (His-Cys-OMe): 272.32. Found by ESI-MS: 273.4 (M+H+). The yields of Cys(Acm)-OMe, Boc-His(Boc)-Cys(Acm)-OMe, and His-Cys-OMe were 99%, 46%, and 90%, respectively, and confirmed by ESI-MS as 204.4 [M+H+], 544.4 [M+H+], and 273.4 [M+H+]. Three different molar ratios of cross-linker and chitosan were designed to produce three types of Chi-CH: Chi-CH A, Chi-CH B, and ChiCH C (Table 1). The ninhydrin results are shown in Table 1 demonstrating the degree of substitution of dipeptide. Chitosan was also measured in the same process as the control group. The data of the degree of substitution of chitosan were subtracted from that of Chi-CH A, B, and C groups. Chi-CH C showed the highest degree of substitution, 4.32%, compared with ChiCH A and Chi-CH B, with a substitution of 2.72% and 3.41%, respectively. Improvement of Buffering Capacity. The titration curve (Figure 1) showed that the Chi-CH C (4.32%) group tolerated more OH- ions than the other two groups. Chitosan also had some buffering capacity between pH 5 and 6; however, the buffering range of chitosan was narrower than that of the histidine-modified chitosan groups. Therefore, Chi-CH C (4.32%) was chosen for use as a gene carrier for the subsequent experiments.

Figure 2. Physical and enzmatic stability of complexes. (a) Chitosan and Chi-CH were dissolved in sodium acetate buffer, pH 5.5, respectively, and mixed with pDNA solution at different A/P ratios.The mixed solutions were incubated at room temperature for 30 min and loaded into the wells of a 1% agarose gel. The gel was stained with SYBR Gold to detect DNA. (b) The complexes prepared at different A/P ratios were incubated with 10 U DNase I at 37 °C for 3 h. The DNA after incubation was extracted using phenol/chloroform/isoamyl alcohol (Nacalai Tesque Inc.) and loaded into the wells of a 1% agarose gel. The gel was stained with SYBR Gold.

Histidine-Modified Chitosan

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Table 2. Particle Size and Zeta Potential of Chitosan/pDNA and Chi-CH/pDNAa Particle Size (unit: nm) pH 5.5

pH 7.4

A/P ratio

Chitosan/pDNA

Chi-D/pDNA

Chitosan/pDNA

Chi-D/pDNA

0.5/1.0 1.0/1.0 2.0/1.0 4.0/1.0 5.0/1.0 10.0/1.0 15.0/1.0 25.0/1.0

37.53 ( 3.82 66.74 ( 7.67 68.42 ( 4.01 51.85 ( 25.85 45.11 ( 26.69 70.26 ( 23.38 74.44 ( 19.58 55.28 ( 33.17

24.12 ( 3.31 39.51 ( 4.15 56.74 ( 7.61 43.49 ( 32.92 46.55 ( 30.14 112.77 ( 3.46 133.00 ( 46.88 144.97 ( 6.48

42.52 ( 4.24 74.72 ( 9.01 225.95 ( 62.57 264.47 ( 55.01 122.8 ( 166.30 135.12 ( 51.29 358.23 ( 229.33 N.D.

30.78 ( 10.78 44.75 ( 4.33 561.96 ( 305.97 30.81 ( 28.31 159.57 ( 10.56 237.50 ( 11.95 91.59 ( 9.48 158.23 ( 32.28

Zeta Potential (unit: mV) pH 5.5

pH 7.4

A/P ratio

Chitosan/pDNA

Chi-D/pDNA

Chitosan/pDNA

Chi-D/pDNA

0.5/1.0 1.0/1.0 2.0/1.0 4.0/1.0 5.0/1.0 10.0/1.0 15.0/1.0 25.0/1.0

-33.6 ( 1.2 -19.3 ( 0.4 31.1 ( 1.2 36.3 ( 0.6 36.9 ( 1.6 33.2 ( 0.6 0.1 ( 0.0 0.7 ( 2.9

-36.0 ( 1.8 -29.1 ( 0.9 -28.8 ( 0.6 36.0 ( 1.1 36.7 ( 1.6 35.3 ( 0.6 35.3 ( 2.5 32.0 ( 5.9

-33.6 ( 0.9 -26.6 ( 1.3 -0.7 ( 0.1 12.50.3 9.0 ( 0.5 15.0 ( 0.8 5.2 ( 0.6 N.D.

-36.5 ( 1.3 -35.3 ( 0.1 -8.1 ( 0.4 5.1 ( 0.3 13.0 ( 2.1 14.0 ( 0.3 16.2 ( 4.4 7.6 ( 0.5

a Chitosan and Chi-CH were dissolved in sodium acetate buffer and mixed with pDNA solution at different A/P ratios. After 30 minutes incubation at room temperature, the particle size was measured by ZetaNanoSizer at pH 5.5 and pH 7.4 (n ) 3).

Physicochemical Characterization of Complexes. The particle size of the complexes (Table 2a) was measured at pH 5.5 and 7.4 in order to evaluate the influence of pH on the dispersion of the complexes. At pH 5.5, both chitosan/pDNA and Chi-CH/pDNA had a particle size lower than 200 nm. At pH 7.4, chitosan/pDNA complexes had a small particle size at an A/P ratio less than 10. However, larger chitosan/pDNA complexes were observed at 15 and 25 A/P ratios, respectively, in contrast to the smaller size of the Chi-CH/pDNA complexes at the same A/P ratio. At A/P 25, ZetaNanoSizer could not measure the particle size of chitosan/pDNA complexes due to the aggregation that occurred at a high A/P ratio, which was beyond the detection limit of the instrument. The zeta potential is shown in Table 2b. At both pH 5.5 and 7.4, chitosan/pDNA and Chi-CH/pDNA complexes had the same zeta potential profile at a A/P ratio less than 10. However, the zeta potential of chitosan/pDNA complexes decreased from A/P 15, while that of Chi-CH/pDNA complexes was constant at A/P 15 and 25. The zeta potential of chitosan/pDNA at A/P 25 in pH 7.4 medium could not be measured due to aggregation. Physical and Enzymatic Stability of Complexes. The physical stability of complexes evaluated by agarose gel electrophoresis (Figure 2a) demonstrated the high stability of both chitosan/pDNA and Chi-CH/pDNA groups, except for chitosan/pDNA at A/P 1. The ability to protect pDNA from enzyme degradation was also evaluated (Figure 2b). Chitosan/pDNA showed some degradation fragments at A/P 1, 2, and 5, while Chi-CH/pDNA showed degradation fragments only at A/P 2. Enhancement of Cellular Uptake. The uptake of fluoresceinlabeled pDNA by HEK293 was measured by flow cytometry at 2, 6, and 12 h (Figure 3). Chitosan/pDNA uptake was low at three time points. Chi-CH/pDNA demonstrated gradually increasing uptake from 2 to 6 h and reached an uptake plateau at 6 h in contrast to the negligible uptake of chitosan/pDNA. Endosomal Escape. Endosomes (pH 5.0-6.0) and lysosomes (pH 5.0-5.5) in HEK293 cells were labeled with Alexa Fluor 488 transferrin conjugate (Figure 4a), LysoTracker Green DND26 (Figure 4b), and LysoSensor Green DND-189 (Figure 4c), respectively. Chitosan/pDNA and Chi-CH/pDNA at A/P 2 and 25 were both examined first in endosome-labeling HEK293 cells

Figure 3. Cellular uptake of complexes. Fluorescein-labeled pDNA (1 µg/mL) complexes prepared by Lipofectamine 2000, chitosan, and Chi-CH were incubated with HEK 293 cells in Opti-MEMI at fixed time points. The extracellular fluorescence was quenched by Trypan blue, and the intracellular fluorescence was measured by flow cytometry. Lipofectamine 2000 was used as a positive control (n ) 3).

(Figure 4a). We found that chitosan/pDNA at A/P 25 and ChiCH/pDNA at A/P 2 did not show obvious intracellular distribution of TMR-labeled pDNA. Therefore, we chose chitosan/ pDNA at A/P 2 and Chi-CH/pDNA at A/P 25 for the following lysosomal escape (Figure 4b,c). Chi-CH/pDNA showed a wide intracellular distribution of TMR-labeled pDNA at A/P 25 compared with a limited and overlapped intracellular distribution of chitosan/pDNA at A/P 2. These results suggested that chitosan/pDNA complexes may be trapped in the endosomes and lysosomes where the pH is lower than 5.5. Effect of Proton Pump Inhibitor on Gene Transfection. Bafilomycin A1 is an H+-ATPase inhibitor, which inhibits acidification in endosomes and lysosomes (27). Generally, bafilomycin A1 is used to indirectly prove that the buffering capacity of PEI increases the endosomal escape rate. This is because bafilomycin A1 treatment inhibits the flow of hydrogen ions from cytosol into vesicles and then inhibits the increase of

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Figure 4. Confocal microscopy of complexes. Chitosan and Chi-CH formed complexes with TMRpDNA in A/P ratios of 2/1 and 25/1. HEK 293 cells were used as models to observe the endosomal escape. Cells were seeded into 12 well plates at 70% confluence. After 24 h incubation, medium of 100 mg/mL Alexa Fluor 488 transferrin conjugate (a), 1 µM LysoTracker Green DND-189 (b), or 1 µM LysoSenser Green DND-26 (c) was added to each well before complex administration. After a 20 to 30 min incubation, chitosan/TMR-pDNA and Chi-CH/TMR-pDNA were added to each well. After a 6 h incubation, the cells were washed by PBS and fixed with 4% paraformaldehyde. The distribution of fluorescence was observed by Nikon confocal microscopy (60× water immersion lens).

osmotic pressure caused by PEI (28). The inhibition of gene expression after bafilomycin A1 treatment (Figure 5) may prove that the hydrogen ion concentration gradient plays an important role in the gene transfection process of Chi-CH/pDNA. After 100 nM bafilomycin A1 treatment, a significant decrease in gene expression was observed in Chi-CH/pDNA and commercially available jetPEI (data not shown), while a slight but insignificant decrease in gene expression could also be recognized in chitosan/ pDNA. Evaluation of Gene Transfection Efficiency. Chitosan/ pDNA and Chi-CH/pDNA complexes at different A/P ratios were prepared and transfected in HEK293 cells for gene expression screening (Figure 6). In the chitosan/pDNA groups, the A/P 2 group showed the highest gene expression

after a 72 h incubation. The A/P 25 group of Chi-CH/pDNA showed the highest gene expression compared with other A/P ratios.

DISCUSSION In this study, we designed His-Cys-OMe after considering three factors. First, the primary amine group in histidine was retained in order to prevent a reduction in nucleic acid binding strength. The positive charge of the primary amino group in histidine could bind to nucleic acids, which would help the formation of chitosan/pDNA complexes. Second, the direct linkage of the amine groups on the chitosan chain and the carboxylic acid group on histidine may reduce the buffering capacity of the histidyl imidazole group. A previous report demonstrated that the buffering range of histidine increased from pH 5-7 to pH 6-8 following conjugation with the acidic group

Histidine-Modified Chitosan

Figure 5. Proton pump inhibition. One hundred nanomolar bafilomycin A1 was added to HEK 293 cells seeded before 24 h into 12-well plates at a density of 1.5 × 104 cells/mL 10 min before complex administration. Complexes and bafilomycin A1 were incubated with the cells for 6 h, and then the medium changed from Opti-MEM I to DMEM(+) and the cells were incubated for another 66 h. The relative light units (RLU) were evaluated by luminometer and calibrated by Bradford protein assay. **p < 0.01 (n ) 3, +Bafilomycin A1 vs -Bafilomycin A1, Student’s t test).

Figure 6. Gene expression of complexes. The gene transfection efficiency of different A/P ratios of chitosan/pDNA and Chi-CH/pDNA was screened by luciferase assay in HEK 293 cells. The RLU were calibrated by Bradford protein assay to obtain an accurate luciferase intensity per milligram protein (n ) 3).

(18). Therefore, chitosan conjugated directly to histidine may increase the pKa of histidine and then decrease the buffering capacity around the pH range of endosomes and lysosomes. Third, the disulfide bond formed between a cross-linker, 2-iminothiolane, conjugated to chitosan and cysteine linked to histidine can be formed at room temperature and in 10% DMSO. This process did not need any other organic solvent or further reagent treatment. No other side product which could disturb the biocompatibility was produced during the synthesis. This reaction could be performed under mild conditions without influencing the biocompatibility of chitosan (25, 29). Materials with a high buffering capacity in endosomes and lysosomes, such as PEI, polylysine, and polyamidoamine, can trap hydrogen ions in endosomes and lysosomes; then, the H+ATPase pump on the membrane of endosomes and lysosomes would introduce more hydrogen ions inside and, finally, disrupt the endosomes and lysosomes because of the high osmotic pressure (30). Endosomal escape can be achieved by the high buffering capacity in the pH range of endosomes and lysosomes. In this study, Chi-CH (4.32%) exhibited the highest degree of substitution of the Chi-CH derivatives (Table 1) and have more

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buffering capacity than chitosan alone in solution (Figure 1). Therefore, Chi-CH (4.32%) was selected for the subsequent analysis and complex preparation. The buffering capacity of Chi-CH was also evaluated in cells by microscopic imaging. LysoSensor Green DND-189 emits a green color when the pH of the vesicles is lower than its pKa (pKa 5.2) corresponding to the lysosomal pH (Figure 4c). This result suggests that the TMRlabeled pDNA carried by Chi-CH was not surrounded by a low pH environment. This might be due to the buffering capacity of Chi-CH neutralizing the low pH in late endosomes or lysosomes and then improving the endosomal escape. In order to evaluate this endosomal escape, we examined the intracellular distribution of pDNA from chitosan/pDNA and Chi-CH/pDNA. Chi-CH/pDNA shows independent scattered red spots (TMRlabeled pDNA) without any overlapping green fluorescence (Alexa Fluor 488 transferrin conjugate in Figure 4a or LysoTracker Green DND-26 in Figure 4b), while chitosan/pDNA at an A/P ratio of 2/1 and 25/1 shows overlapping red and green fluorescence and aggregation of red fluorescence on the cell membrane, respectively. This result suggests that Chi-CH/pDNA escaped from endosomes or lysosomes more efficiently than chitosan/pDNA. The endosomal escape ability of Chi-CH/pDNA would be due to the buffering capacity provided by histidine. In addition, the gene expression of Chi-CH/pDNA with bafilomycin A1 treatment was lower than that without bafilomycin A1 treatment. This phenomenon could be explained by the higher buffering capacity of Chi-CH compared with chitosan. With these results taken into consideration, histidine modification could increase the endosomal escape of chitosan complexes efficiently by increasing the buffering capacity of chitosan. The high cellular uptake was also an important factor for improving the gene transfection efficiency of Chi-CH/pDNA. Chitosan/pDNA shows negligible mean fluorescence intensity in cells even after a 12 h incubation (Figure 3). In combination with the result in Figure 4, which showed aggregation on the cell membrane, these results suggest that the low gene expression of chitosan/pDNA was mainly due to the slow pDNA release from the aggregates on the cell membrane. Moreover, the particle size of Chi-CH/pDNA was smaller than that of chitosan/pDNA at A/P ratios higher than 15 under neutral conditions (Table 2). The zeta potential of chitosan/pDNA also indicated a decreasing trend of chitosan/pDNA at a A/P ratio of 15 under neutral conditions (Table 2). These results suggest that chitosan/pDNA may encourage aggregation under neutral conditions. At pH 5.5, although the particle size of chitosan/ pDNA did not increase at A/P ratios higher than 15, the zeta potential also shows the decreasing trend. Zeta potential is related to the electrophoretic mobility which was determined by ZetaNanoSizer. At A/P ratio higher than 15, the increasing viscosity of chitosan (31) and partial aggregation of chitosan/ pDNA may affect the decrease of zeta potential. As far as cellular uptake is concerned, Chi-CH/pDNA shows nearly an 8-fold higher cellular uptake than chitosan/pDNA (Figure 3). The increase in the solubility after histidine conjugation, from nearly 0 mg/mL to 10 mg/mL under neutral conditions (data not shown), also provided a possible explanation for the higher cellular uptake. In several reports, the degree of aggregation of chitosan/pDNA has been shown to be reduced by increasing the water solubility of chitosan, such as modification by hydrophilic groups (13) and adjusting the molecular weight of chitosan (6, 9, 32). Therefore, the improvement in the cellular uptake of Chi-CH/pDNA might be partly attributed to the higher solubility of Chi-CH compared with chitosan. In order to solve the problem of the slow endosomal escape rate during the process of drug or gene delivery, other reports have described how chitosan could also be modified with moieties which have a buffering capacity, including imidazole

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(33) and N-R-acetyl-L-histidine (34). Our results are in agreement with these reports. The modification of imidazole proved that the increase in buffering capacity can increase the gene transfection efficiency of chitosan (33). The modification of N-Racetyl-L-histidine on glycol chitosan also allowed the endosomal escape of paclitaxel (34). However, the acetylation on the N terminal end of histidine may increase the pKa of the imidazole ring and then shift the buffering range of histidine to a higher pH. For example, the pKa of histidine increases to 7-8 adjacent to an acidic amino acid (18), and the pKa of N-R-acetyl-Lhistidine is 7.2 (35). The shift of buffering range may reduce the buffering capacity of histidine in endosomes and lysosomes. In our design, the primary amino group (pKa 9.2) and the imidazole ring (pKa 6.0) could be prevented from basic shift of pKa because we avoid chemical modification at the N terminal end of histidine.

CONCLUSION We conjugated histidine to chitosan and increased the gene transfection efficiency of chitosan successfully by increasing the buffering capacity in the pH range of endosomes and lysosomes without affecting the integrity of the complexes. These results suggest that histidine helped the uptake of pDNA by cells and increased the efficiency of escape from endosomes and lysosomes.

ACKNOWLEDGMENT This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and by Research on Publicly Essential Drugs and Medical Devices in Japan Health Sciences Foundation.

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