Agmatine-Containing Bioreducible Polymer for Gene Delivery

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Agmatine-containing bioreducible polymer for gene delivery systems and its dual degradation behavior

Ji-yeong Choi,†,a Kitae Ryu,†,a Gyeong Jin Lee,† Kyunghwan Kim,† and Tae-il Kim*,†,‡

†

Department of Biosystems & Biomaterials Science and Engineering, College of Agriculture

and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea ‡

Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-

ro, Gwanak-gu, Seoul 151-921, Republic of Korea

Corresponding author: Prof. Tae-il Kim Department of Biosystems & Biomaterials Science and Engineering College of Agriculture and Life Sciences, Seoul National University 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea Tel: 82-2-880-4636 Fax: 82-2-873-2285 e-mail: [email protected]

a

These authors contributed to this work equally.

1 ACS Paragon Plus Environment

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Abstract Agmatine-containing

bioreducible

polymer,

poly(cystaminebisacrylamide-agmatine)

(poly(CBA-AG)) was synthesized for gene delivery systems. It could form 200-300 nm sized and positively charged polyplexes with pDNA, which could release pDNA in reducing environment due to the internal disulfide bonds cleavage. poly(CBA-AG) also showed a spontaneous degradation behavior in aqueous condition in contrast to the backbone polymer, poly(cystaminebisacrylamide-diaminobutane) (poly(CBA-DAB)) lacking guanidine moieties, probably due to the self-catalyzed hydrolysis of internal amide bonds by guanidine moieties. The cytotoxicity of poly(CBA-AG) was cell-dependent but minimal. poly(CBA-AG) exhibited highly enhanced transfection efficiency in comparison with poly(CBA-DAB) and even higher transfection efficiency than PEI25k. However, cellular uptake efficiency of the polyplexes didn’t show positive correlation with the transfection efficiency. Confocal microscopy observation revealed that pDNA delivered by poly(CBA-AG) was strongly accumulated in cell nuclei. These results suggested that high transfection efficiency of poly(CBA-AG) may be derived from the efficient pDNA localization in cell nuclei by guanidine moieties and that the polyplexes dissociation via self-catalyzed hydrolysis as well as disulfide bonds cleavage in cytosol also may facilitate the transfection process. Fianlly, poly(CBA-AG)/pJDK-apoptin polyplex showed a high anticancer activity induced by apoptosis, demonstrating a potential of poly(CBA-AG) as a gene carrier for cancer gene therapy.

Keywords: Bioreducible polymer, Gene delivery, Agmatine, Self-catalyzed hydrolysis, Nuclear localization ability, Apoptin


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1. Introduction Plenty of cationic bioreducible polymers, which possess internal disulfide bonds, have been developed for non-viral gene delivery systems within last two decades.1-3 They show relatively high stability in extracellular aqueous environment in comparison with hydrolytic polymers retaining ester, amino ester, or phosphoester bonds4-6 and also show selective degradability in cytosol containing high level of reducing agents such as glutathione (0.5-10 mM in cytosol).7,8 These unique properties of bioreducible polymers could be strong advantages for gene delivery systems, such as low cytotoxicity and controllable pDNA release from the polyplex by avoiding the accumulation of high molecular weight polymers inside cells and facilitating polyplex dissociation in cytosol. Therefore, many bioreducible polymers with diverse chemical structures, showing efficient transfection efficiency and low cytotoxicity have been applied for therapeutic gene delivery successfully.9 Moreover, additional chemical modifications to bioreducible polymers have been performed in order to improve the transfection efficiency and to assign specific biological functionalities such as targeting ligand moieties.10-13 Among them, introduction of cellular penetrating moieties such as arginine conjugation or guanidinylation has been proven as a simple and efficient strategy. Arginine or guanidine moieties are found to be rich in cellular penetrating peptides (CPPs) such as Tat peptide or oligoarginine and are thought to play important roles for intracellular transportation of CPPs.14-16 It was reported that arginine-conjugated bioreducible polymer (ABP)17 and guanidinylated bioreducible polymer (GBP)18 showed highly enhanced transfection efficiency with low cytotoxicity in comparison with unmodified bioreducible backbone polymer, poly(CBA-DAH). However, cellular uptake of the polyplexes with cellular penetrating moieties was not improved in these studies and so it was suggested that pDNA localization in cell nuclei by guanidine moieties as well as reductive


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degradation of bioreducible polymers in cytosol could function importantly for the enhanced transfection.17-19 Agmatine (4-aminobutylguanidine) is one of biogenic amines which initially attracted attention as an endogenous ligand at imidazoline receptors and α2-adrenoceptors.20 It is also known to mediate many physiological functions as a neurotransmitter of central nervous system and to be involved in cell growth and proliferation.21 Agmatine is synthesized via decarboxylation of L-arginine by mitochondrial enzyme, arginine decarboxylase in mammalian cells22 and still possesses a guanidine moiety which enables to condense pDNA or to act as cell penetrating functionality. Therefore, several works reported that the introduction of agmatine moiety to polyamidoamines23,24 or fatty acid modified dextran25 for efficient gene delivery systems. In this work, we synthesized agmatine-containing bioreducible polymer, poly(CBA-AG) which is synthesized by Michael addition of cystaminebiscarylamide and diaminobutane. Interestingly, spontaneous degradation of poly(CBA-AG) was observed in normal aqueous condition and this degradation behavior was examined in bioreducible polymer for the first time. Other physico-chemical characterization about pDNA condensing ability, degradability in reducing condition, average sizes and Zeta-potential values of the polyplexes were performed. Cytotoxicity and transfection efficiency, and cellular uptake of polyplexes were also examined in cells. Intracellular trafficking of the polyplexes was observed in fluorescence-stained cells by confocal microscopy and the transfection mechanism of poly(CBA-AG) was suggested. Finally, cancer cell-killing effect of poly(CBA-AG)/pJDKapoptin polyplex was evaluated to indentify the potential for cancer gene therapy.

2. Materials and Methods


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2.1. Materials. Polyethylenimine (PEI, 25 kDa), N,N’-cystaminebisacrylamide (CBA), 1Hpyrazole-1-carboxamidine hydrochloride (1HPC), N,N-diisopropylethylamine (DIPEA), trifluoroacetic

acid

(TFA),

triisopropylsilane,

3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide (MTT), and agarose were purchased from Sigma–Aldrich (St. Louis, MO). N-(tert-butoxycarbonyl)-1,4-diaminobutane (N-Boc-DAB) was purchased from TCI Co., Ltd. (Japan). Dithiothreitol (DTT) was purchased from Biosesang. INC (Korea). The plasmid DNA, pCN-Luci containing a firefly luciferase reporter gene was amplified in Escherichia coli DH5α and isolated by Nucleobond® Xtra Midi kit (Macherey-Nagel, Germany). Luciferase assay system and reporter lysis buffer were purchased from Promega (Madison, WI). Fetal bovine serum (FBS), 0.25% Trypsin-EDTA, Dulbecco's phosphate buffered saline (DPBS), Dulbecco's modified Eagle's medium (DMEM), Hoechst 33342, LysoTracker Red DND-99, and YOYO-1 Iodide (1 mM solution in DMSO) were purchased from Invitrogen (Carlsbad, CA). BCATM protein assay kit was purchased from PIERCE (Rockford, Il). Apoptin gene-encoding plasmid DNA, pJDK-apoptin pDNA26 is a kind gift from Prof. Jong-Sang Park. All other chemicals were purchased and used without any further purification.

2.2. Synthesis and characterization of poly(CBA-AG). According to the procedures reported earlier27, bioreducible backbone polymer, poly(CBA-DAB) was synthesized by Michael reaction of equivalent moles of N-Boc-DAB and CBA in MeOH/H2O solution (9:1, v/v) in the absence of light under N2 atmosphere at 60 oC for 5 days. Then, 10% mole of NBoc-DAB was added to the reaction mixture to consume unreacted acrylamide groups of products and the reaction was maintained for 24 h. After precipitation with diethyl ether, the Boc groups were removed by the strong acid reagent solution (TFA: triisopropylsilane: H2O


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= 95: 2.5: 2.5) at ice bath for 30 min in order to expose primary amines. After additional precipitation with diethyl ether, the product was dialyzed against ultra-pure water with dialysis membrane (MWCO = 2000) for 24 h, followed by lyophilization to leave poly(CBADAB). Then, the primary amines of poly(CBA-DAB) were guanidinylated with 5 equivalent moles of 1HPC and DIPEA in water at room temperature for 1 day to convert the pendant DAB moiety to agmatine, finally synthesizing poly(CBA-AG). After dialysis against ultra-pure water overnight, poly(CBA-AG) was lyophilized before use for analysis. The syntheses of resulting polymers were confirmed by 1H NMR (400 MHz, D2O). The synthesis scheme of poly(CBA-AG) was displayed in Figure 1. The molecular weight of poly(CBA-AG) was measured by gel permeation chromatography (GPC: YL-9100, Young Lin Instrument, Korea). Polyethyleneglycols with various molecular weights were used as standards. The polymer was dissolved at a concentration of 10 mg/mL. The assay was run on Ultrahydrogel 250 column with 1% formic acid as an eluent at 1.0 mL/min of flow rate.

2.3. Degradation of poly(CBA-AG) in aqueous condition. Degradation profile of poly(CBA-AG) in aqueous medium was investigated by measuring the molecular weights of the polymer samples. poly(CBA-DAB) was used as a control. The polymer solutions (PBS buffer (pH 7.4), 10 mg/mL) were incubated at 37 oC. Then, aliquots of the polymer solutions collected at pre-determined time ranging from 5 h to 96 h were analyzed by GPC.

2.4. Agarose gel electrophoresis. Agarose gel electrophoresis was performed in order to examine pDNA condensation ability of poly(CBA-AG) via electrostatic interaction. Polyplexes were prepared in Hepes buffer saline (10 mM Hepes, 1 mM NaCl, pH 7.4) at


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various weight ratios. Agarose gel (0.7%, w/v) containing ethidium bromide DNA gel stain solution (0.5 µg/mL) was prepared in TAE (Tris-Acetate-EDTA) buffer. After 30 min of incubation at room temperature, the polyplex solutions were electrophoresed for 12 min at 100 V (Mupid-2plus, Takara Bio Inc., Japan). In addition, the identical polyplexes were incubated in the presence of 2.5 mM DTT for 30 min at 37 oC and electrophoresed in order to investigate the behavior of poly (CBA-AG) polyplexes in reducing environment. The pDNA bands were visualized by UV illuminator (ChemiDoc XRS+ gel documentation system, BioRad, Hercules, CA).

2.5. PicoGreen assay. PicoGreen assay was performed using a Quant-iT™ PicoGreen® kit (invitrogen) to examine the polymer degradation behavior by detecting the dissociated pDNA from the polyplex. poly(CBA-DAB) and poly(CBA-AG) were used. After formation of polyplex in TE buffer at a weight ratio of 10, each polyplex was incubated at 37 oC for predetermined time. Then, PicoGreen reagent (TE buffer) was added to the polyplex solution and incubated for 4 min. Fluorescence was measured with an excitation wavelength of 480 nm and emission wavelength of 520 nm using a microplate reader (Synergy H1, BioTek, USA). In addition, the identical polyplexes were incubated in presence of 2.5 mM DTT at 37 o

C and analyzed by PicoGreen assay after various incubation times in order to investigate the

degradation behavior of polyplex in reducing environment.

2.6. Average sizes and Zeta-potential values measurement of poly (CBA-AG) polyplexes.

The average sizes and Zeta-potential values of poly (CBA-AG) polyplexes

were measured by Zeta-sizer Nano ZS (Malvern Instruments, UK) with He-Ne laser beam (633 nm) at 25 oC. Polyplexes (5 µg pDNA) were prepared in ultra-pure water at various weight ratios ranging from 0.5 to 30. After 30 min of incubation, the polyplex solutions were


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diluted to 1 mL before measurements. Average particle sizes and Zeta-potential values were measured 3 times.

2.7. Transmission electron microscopy (TEM) observation. 10 µL of poly(CBA-AG) polyplex solutions (0.5 µg pDNA) were prepared at weight ratios of 10 and 30. They were adsorbed on TEM copper grid plates and then the samples were stained with uranyl acetate solution for 30 s. After absorption of residual solutions, the polyplex images were visualized by TEM (JEM1010, JEOL, Japan) with an accelerating voltage of 80 kV.

2.8. Cell culture. Human cervical adenocarcinoma cells (HeLa), mouse neuroblastoma cells (Neuro 2a), and Human lung adenocarcinoma epithelial cells (A549) were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in humidified atmosphere containing 5% CO2 at 37 °C.

2.9. Cytotoxicity. Cytotoxicity of the polymers and polyplexes was measured by MTT assay in HeLa, Neuro 2a, and A549 cells. Cells were seeded in 96-well cell culture plates at a density of 1 x 104 cells/well. When achieving 70-80% of confluency after 24 h, the cells were exposed to 100 µL of polymer solutions or polyplex solutions (0.1 µg pCN-Luci) with various concentrations in serum-free medium for 4 h. After exchange of medium with fresh medium containing 10% FBS, the cells were further incubated for 24 h before assay. Then, the cells were treated with MTT solution (2 mg/mL in DPBS) for 2 h at 37 °C. After removing each medium carefully, the formazan crystal formed by proliferating cell was dissolved in 150 µL of DMSO. The absorbance was measured at 570 nm using a microplate


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reader. Results were presented as relative cell viabilities (RCV, percentage values relative to value of untreated control cells). All experiments were performed in quadruplicate.

2.10. Transfection experiments in vitro. Transfection efficiency was examined by luciferase transgene expression assay in HeLa, Neuro 2a, and A549 cells. The cells were seeded in 24-well cell culture plates at a density of 5 x 104 cells/well in culture medium. When the cells achieved 70-80% of confluency after 24 h, the media were exchanged with DMEM for the assay in serum-free condition or with DMEM (10% FBS) for the assay in serum condition, respectively. Then, the cells were treated with polyplex solutions (0.5 µg pDNA) for 4 h. PEI25k polyplex (weight ratio=1) was used as a control. After exchange with fresh medium containing 10% FBS, the cells were further incubated for 2 days. For analysis, the growth medium was removed and cells were rinsed with DPBS and shaken for 30 min at room temperature with 120 µL of Reporter Lysis Buffer to obtain cell lysate. Luciferase activities of cell lysates were measured by using luciferase assay reagents on a microplate reader. A protein quantification assay was performed using a BCATM Protein Assay Reagent Kit to measure total amount of cellular proteins. The final results were presented in terms of RLU/mg cellular protein. All experiments were performed in triplicate.

2.11. Cellular uptake of polyplexes. HeLa and Neuro 2a cells were seeded in 6-well cell culture plates at a density of 2 x 105 cells/well in culture medium, respectively. Having achieved 70-80% of confluency after 24 h, each medium was exchanged for fresh serum-free medium. pDNA labeled with YOYO-1 Iodide (1 molecule of the dye per 50 base pairs of the nucleotide) was prepared. The cells were treated with poly(CBA-AG) polyplex solutions (1 µg pDNA) at various weight ratios (10, 30, and 50) for 4 h at 37 °C. PEI25k polyplex was used as a control. Then, each medium was removed and the cells were washed two times with


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ice-cold DPBS. After trypsinization, the cells were re-suspended in DPBS. The cellular uptake of fluorescence-labeled polyplexes was examined by using the BD FACScan analyzer (Becton Dickinson, San Jose, CA) at a minimum of 1 ×104 cells gated per sample. Analysis was performed by using Becton Dickinson CellQuest software. Data were processed by using Flowing software.

2.12. Intracellular trafficking of polyplexes. Intracellular trafficking of polyplexes was observed by using confocal laser scanning microscope (CLSM). HeLa cells were seeded in confocal imaging dishes at a density of 8 x 104 cells/dish. After 24 h of incubation, the media were exchanged with fresh DMEM. The cells were subsequently treated with polyplexes solutions (0.5 µg pDNA labeled by YOYO-1 Iodide, PEI25k: weight ratio=1, poly(CBADAB) and poly(CBA-AG): weight ratio=20). The cells were washed with DPBS after 4 h of incubation and stained by Hoechst 33342 (10 µg/mL) and LysoTracker Red (50 nM) for 15 min. After exchange of fresh DMEM, the cell images were obsrved by CLSM (Leica TCS SP8X Gated STED, Germany) and processed by LAS AF Lite (Leica Application Suite, Advanced Fluorescence Lite) program.

2.13. Accessment of anticancer effect by MTT assay. poly(CBA-AG) polyplexes (1 µg of pJDK-apoptin pDNA) were prepared at various weight ratios and treated to A549 cells according to the transfection method as described above (Section 2.10). PEI25k polyplex (weight ratio=1) and poly(CBA-DAB) polyplexes with various weight ratios were also used as controls. After 2 days of incubation, cancer cell killing effect by apoptin gene delivery was examined by MTT assay. Results were presented as relative cell viabilities. All experiments were performed in triplicate.


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2.14. Reverse transcription polymerase chain reaction (RT-PCR). RT-PCR analysis was performed for the identification of apoptin expression in A549 cells. PEI25K, poly(CBADAB), and poly(CBA-AG) polyplex (1 µg pJDK-apoptin pDNA) solutions were prepared at various weight ratios and treated to the cells as described above (Section 2.10). After 2 days of transfection, total RNA was isolated from the cells with TRIzol® RNA isolation reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RT-PCR analysis was performed using OneStep RT-PCR kit (QIAGEN, Hilden, Germany). Primers for RTPCR were purchased from BIONEER (Korea). Glyceraldehydes 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used as a control. Primer sequences are shown below. Apoptin Forward: 5’-CCC GAA TTC ATG AAC GCT CTC-3’, Apoptin Reverse: 5’-GCC CTC TAG ATC ACA GTC TTA TAC GC-3’, GAPDH Forward: 5’-GAC CTG CCG TCT AGA AAA AC-3’, GAPDH Reverse: 5’-TTG AAG TCA GAG GAG ACC AC-3’ After amplification, agarose gel electrophoresis were performed with 2% agarose gel for 20 min and amplified cDNA for apoptin and GAPDH mRNA were visualized by UV illuminator.

2.14. Annexin V/propidium iodide (Annexin V/PI) apoptosis assay. Annexin V/PI apoptosis assay was performed in order to examine the anticancer effect of apoptin gene delivery mediated by apoptosis. A549 cells were seeded in 6-well cell culture plates at a density of 2 x 105 cells/well in culture medium. Having achieved 70-80% of confluency after 24 h, each medium was exchanged for fresh serum-free medium. The cells were treated with poly(CBA-AG) polyplex solutions at various weight ratios for 4 h at 37 °C. PEI25k polyplex (weight ratio=1) and poly(CBA-DAB) polyplexes (weight ratio=100) were also used as controls. After exchange with fresh medium containing 10% FBS, the cells were further incubated for 2 days. Then, the cells were washed with DPBS and stained with Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit (Invitrogen). Stained cells were analyzed by using


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the BD FACScan analyzer at a minimum of 1 ×104 cells gated per sample. Analysis was performed by using Becton Dickinson CellQuest software. Data were processed by using Flowing software.

3. Result and Discussion 3.1. Synthesis and characterization of poly(CBA-AG). poly(CBA-DAB)27, which is composed of N,N’-cystaminebisacrylamide (CBA) and diaminobutane (DAB), was employed as a backbone polymer for the synthesis of agmatine-containing bioreducible polymer, poly(CBA-AG). Diaminobutane side chain of poly(CBA-DAB) can be converted to agmatine after guanidinylation of an exposed terminal primary amine. Briefly, poly(CBA-DAB) was synthesized by Michael reaction of CBA and N-Boc-DAB at equivalent molar ratio. Then, the exposed primary amines of poly(CBA-DAB) after removal of Boc groups were guanidinylated by using 1HPC and DIPEA, leading to the synthesis of poly(CBA-AG). The synthesis of poly(CBA-AG) was confirmed by 1H NMR as follows (Figure 1). G means guanidine groups of agmatine side chains.

Figure 1. Synthesis scheme of poly(CBA-AG) and 1H NMR spectra of poly(CBA-DAB) and poly(CBA-AG). (i) MeOH:H2O=9:1, (v/v, without light under N2, 60 oC, 5 days), (ii) TFA:triisopropylsilane:H2O=95:2.5:2.5 (v/v, 0 oC, 30 min).


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1

poly(CBA-DAB);

H

NMR

(D2O):

δ (NH2CH2CH2CH2CH2N-)=1.57-1.68,

δ

(-

NCH2CH2CONHCH2CH2SS-)=2.45, δ (NH2CH2CH2CH2CH2N-)=2.55, δ (-CH2SSCH2-)= 2.84, δ (-NCH2CH2CONHCH2CH2SS-)=2.88, δ (NH2CH2CH2CH2CH2N-)=3.02 δ (NCH2CH2CONHCH2CH2SS-)=3.54. poly(CBA-AG);

1

H

NMR

(D2O):

δ(G-NCH2CH2CH2CH2N-)=1.54-1.62,

δ

(-

NCH2CH2CONHCH2CH2SS-)=2.42, δ (G-NCH2CH2CH2CH2N-)=2.49, δ (-CH2SSCH2-)= 2.80, δ (-NCH2CH2CONHCH2CH2SS-)=2.84, δ (G-NCH2CH2CH2CH2N-)=3.18, δ (NCH2CH2CONHCH2CH2SS-)=3.50. After guanidinylation of DAB primary amines, the intensity of protons next to primary amines (proton a in Figure 1, δ=3.02) was greatly diminished and shifted downfield (proton b in Figure 1, δ=3.18), which come from the protons next to guanidine groups. According to the comparison of intensities between these proton peaks, it was found that about 97.5% of primary amines in poly(CBA-DAB) was converted to guanidines. From this result, it was concluded that the synthesis of poly(CBA-AG) was performed successfully. The weight average-molecular weight (Mw) of poly(CBA-AG) was measured to be 6.74 kDa by gel permeation chromatography (GPC) and its PDI value was 1.85.

3.2. Degradation of poly(CBA-AG) in aqueous condition. In general, it is known that bioreducible polymers composed of polyamines and cystaminebisacrylamide are not degraded well in aqueous medium of normal condition, which suggests their good stability in extracellular environment and selective degradability in reducing environment such as cytosol. However, we observed the spontaneous degradation of poly(CBA-AG) in aqueous medium of normal condition and so investigated the degradation of the polymer by GPC. Unmodified backbone polymer, poly(CBA-DAB) was used as a control.


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After incubation of polymers in PBS buffer during predetermined time period, the polymer solutions were analyzed by GPC and the result was shown in Figure 2A. The average Mw of degraded polymer fragments in each sample was calculated as a mean value of each fragment peak Mw multiplied by the corresponding peak area (%). Average Mw of poly(CBA-DAB) showed no significant change, maintaining about 6 kDa/mole of Mw even after 96 h of incubation, which means poly(CBA-DAB) is not degraded and shows high stability in aqueous environment. In fact, GPC peak of poly(CBA-DAB) remained completely intact during the whole incubation time (Figure S1). However, in the case of poly(CBA-AG), several peaks appeared during incubation, which demonstrated that poly(CBA-AG) was degraded into several fragments. Average Mw of poly(CBA-AG) fragments was decreased abruptly to 2.7 kDa/mole only after 5 h of incubation and gradually decreased to less than 1 kDa/mole after 48 h of incubation. This result indicates that poly(CBA-AG) can be degraded in even normal aqueous environment. Considering the difference of chemical structure between poly(CBA-DAB) and poly(CBA-AG), it is deduced that this may be induced via the cleavage of backbone by self-catalyzed hydrolysis of guanidine moieties. Other characterizations for the polymer degradation were performed to examine the degradation mechanism. Primary amines and thiols of poly(CBA-AG) degradation samples were quanitified in order to indentify the degraded bonds by fluorescamine assay and Ellman’s assay, respectivley. Primary amines will be exposed if amide bonds are cleaved and thiols will be exposed if disulfide bonds are cleaved during incubation in aqueous condition. In Figure S2, primary amines of poly(CBA-AG) degradation samples were increased to 6 folds higher amounts after 72 h of incubation in comparison with initial primary amines of intact poly(CBA-AG). In Figure S3, thiols of poly(CBA-AG) degradation samples didn’t show any significant changes regardless of incubation time. Therefore, it is concluded that


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internal amide bonds of poly(CBA-AG) may be degraded during incubation in aqueous medium, not disulfide bonds.

Figure 2. (A) Average Mw of polymer fragments depending on incubation time in PBS buffer (pH 7.4). (B) Hypothetic scheme for poly(CBA-AG) degradation by self-catalyzed hydrolysis of guanidine moiety. Dotted line: hydrogen bond, arrow: direction of electron transfer.

Degradation profile of poly(CBA-AG) in the presence of guanidinylation reagents was examined in order to identify the degradation during guanidinylation process and the effect of the reagents on the degradation of poly(CBA-AG) (Figure S4). Degradation rate of poly(CBA-AG) was decreased with the increase of guanidinylation reagents. Therefore, it is elucidated that degradation of poly(CBA-AG) during guanidinylation in aqueous solution can be suppressed in the presence of 1HPC and DIPEA and that guanidine moiety of 1HPC can interfere the catalytic activity of agmatine guanidine moiety by competitive interaction with internal amide bonds.


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In order to investigate the concentration effect on the degradation, poly(CBA-AG) samples with two different concentrations (0.1 mg/mL and 10 mg/mL, respectively) were prepared and the degradation profiles were analyzed by GPC. Interestingly, poly(CBA-AG) sample with low concentration showed faster degradation than that with high concentration (Figure S5). This result means that intramolecular reaction may be superior to that by intermolecular reaction for degradation of poly(CBA-AG) and that the cleavage of poly(CBA-AG) amide bond by adjacent guanidine moiety may be interfered by guanidine moieties of other polymer chains at high concentration probably due to the competitive interaction as mentioned above. In order to examine the pH effect on the degradation, poly(CBA-AG) solutions with two different pH (pH 4.5 and pH 8.0) were prepared and the degradation profiles were analyzed by GPC (Figure S6). In acidic condition (pH 4.5, sodium acetate buffer), no degradation behavior of poly(CBA-AG) was observed. However, rapid decrease of poly(CBA-AG) molecular weight was observed at pH 8.0 (sodium phosphate buffer). It is thought that protonation of carbonyl oxygen at acidic pH may interrupt the initiative interaction between carbonyl oxygen and guanidine moiety via proper hydrogen bonds, which can suppress the subsequent hydrolysis reaction. In addition, the molecular weight change of backbone polymer, poly(CBA-DAB) in the presence of free agmatine molecules (0.1X-10X) was also analyzed in order to examine the catalytic activity of guanidine moiety (Figure S7). The fastest degradation of poly(CBADAB) was found in 0.1X agmatine condition. 1X and 10X agmatine condition just induced the mild degradation (degradation rate: 1X>10X). Therefore, it is thought that guanidine moiety can conduct the catalytic hydrolysis of poly(CBA-DAB) amide bond and this may be interfered by adjacent other guanidine groups at high concentration, probably due to the competitive interaction, as already suggested above. This result supports the intramolecular mechanism of degradation.


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Actually, guanidine moiety has been used for as an organocatalyst for many reactions28,29 and it was proposed that the mechanism for ring opening polymerization of polyesters by guanidine catalyst may be based on hydrogen bonding to the catalyst.30 According to this cleavage mechanism of cyclic ester by guanidine, we present the hypothetic mechanism of guanidine-catalyzed amide bond cleavage in Figure 2B. Protonated guanidine moiety will form di-hydrogen bonds with carbonyl oxygen in the adjacent amide bond, which can make carbonyl carbon more electrophilic and induce faster nucleophilic attack of a water molecule. After formation of tetrahedral structure, oxonium ion will be converted to hydroxyl group by acid/base reaction of secondary amine. Then, the secondary amine will be converted to ammonium ion, a good leaving group and electrons of alkoxide ion will push out the ammonium ion, cleaving C-N bond. Jine et al. also suggested the possibility of amide bond cleavage by adjacent guanidine groups in guanidinylated poly-L-lysine at pH 5.0 but the detailed mechanism about the degradation was not discussed.31 In this work, for the first time, we suggest that bioreducible polymer, poly(CBA-AG) can be degraded via cleavage of internal amide bonds by self-catalyzed hydrolysis of guanidine moieties in normal aqueous condition.

3.3. pDNA condensation by poly(CBA-AG). Agarose gel electrophoresis analysis was carried out in order to examine pDNA condensing ability of poly(CBA-AG) via electrostatic interaction. poly(CBA-AG) polyplexes were also electrophoresed in the presence of 2.5 mM DTT, a well-known reducing agent, in order to confirm the biodegradability of poly(CBAAG) in intracellular reducing environment. In Figure 3A, it was found that poly(CBA-AG) could retard pDNA from a weight ratio of 2 in the absence of DTT, indicating that poly(CBA-AG) can condense pDNA to positively


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charged polyplex particles stably via electrostatic interaction at a low weight ratio. However, pDNA release was observed even at a weight ratio of 20 in the presence of DTT (Figure 3B). This result means that poly(CBA-AG) can be degraded in reducing environment due to the cleavages of disulfide bonds within its backbone and release pDNA from polyplexes simultaneously.

Figure 3. pDNA condensation by poly(CBA-AG). Agarose gel electrophoresis results of poly(CBA-AG) polyplexes (A) without and (B) with 2.5 mM DTT. Numbers mean the weight ratios of polyplexes. Picogreen assay results of poly(CBA-AG) polyplexes (C) without and (D) with 2.5 mM DTT


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In order to examine the degradation of the polyplexes in aqueous medium, PicoGreen assay was also performed. In Figure 3C, poly(CBA-AG) polyplex showed very low fluorescence values of PicoGreen reagent even after 20 h of incubation, meaning no significant release of pDNA from the polyplex. A control polyplex, poly(CBA-DAB) polyplex also showed the low fluorescence values. The identical experiments were also performed in the presence of DTT (Figure 3D). In this case, both poly(CBA-AG) and poly(CBA-DAB) polyplexes showed high fluorescence values of PicoGreen reagents even after 30 min of incubation, meaning the significant release of pDNA from the polyplex due to the degradation of the polymers. Therefore, it is thought that self-catalytic degradation of poly(CBA-AG) is inhibited in the polyplex form probably due to the restricted reactivity and motility of guanidine moiety by electrostatic interaction or binding with pDNA, not like free poly(CBA-AG) polymer form. For these results, it is anticipated that poly(CBA-AG) polyplex can release pDNA only in cytoplasm after cellular uptake, leading to the efficient transfection by bioreductioncontrolled manner. Taken together with the above results, it is concluded that poly(CBA-AG) possesses dual biodegradability, which may show specific behavior for gene delivery systems.

3.4. Characterization of poly(CBA-AG) polyplexes. Zeta-potentials and average diameters of poly(CBA-AG) polyplexes were measured by using Zeta-sizer. It was reported that positively charged polyplexes can interact with negatively charged cellular membrane allowing of enhanced uptake into cells and their proper sizes are also needed for efficient cellular uptake.32,33 As shown in Figure 4A, Zeta-potential value of the polyplex was measured to be negative charge values (-18.5 mV) at a weight ratio of 0.5, indicating that negatively charged pDNA was not condensed completely by poly(CBA-AG) at that ratio. Then, at higher weight ratios


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over 1, potential values turned to be positive and showed almost 40 mV values at weight ratios ranging from 10 to 30, meaning the formations of stable polyplexes condensed by cationic poly(CBA-AG). It is consistent with the agarose gel electrophoresis in which pDNA was condensed by poly(CBA-AG) from a weight ratio of 2. In the case of polyplex sizes, poly(CBA-AG) displayed the highest values (560 nm) at a weight ratio of 1 and the sizes were found to be abruptly decreased to 200-300 nm with the increase of polyplex weight ratios. It is notable that at a weight ratio of 1 showing almost zero value (0.6 mV) of Zetapotential, large aggregates of polyplexes were formed probably due to the hydrophobic interactions between electrically neutralized polyplexes. Sizes of polyplexes were dramatically decreased at higher weight ratios due to the formation of positively charged polyplexes with addition of the cationic polymer. poly(CBA-DAB) polyplexes also showed similar positive Zeta-potential values and average sizes (200-300 nm) with poly(CBA-AG) polyplexes (Figure S8), suggesting that there are no significant differences of physicochemical properties between these two polyplexes. These results demonstrate that poly(CBA-AG) could form positively charged and nanosized polyplex particles with pDNA, which have suitable values for efficient cellular uptake of polyplexes. In addition, it was found that the polyplex formation behavior of poly(CBAAG) regarding size and Zeta-potential was dependent on the weight ratios of polyplexes which are closely related with electrostatic property of polyplexes. Morphologies of poly(CBA-AG) polyplexes were also observed by TEM. It was shown that poly(CBA-AG) polyplexes appeared as spherical shapes with sizes less than 200 nm at weight ratios of 10 and 30 in Figure 4. The sizes were found to be smaller than the values obtained by Zeta-sizer. It is thought that polyplexes sizes observed by TEM in dried condition would be smaller than hydrodynamic sizes of the same polyplexes measured by Zeta-sizer in aqueous solution.


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Figure 4. (A) Average size and Zeta-potential value measurements of poly(CBA-AG) polyplexes. TEM images of poly(CBA-AG) polyplexes at weight ratios of (B) 10 and (C) 30

3.5. Cytotoxicity. Cytotoxicity of poly(CBA-AG) was assessed by MTT assay, as shown in Figure 5. HeLa cells, Neuro 2a cells, and A549 cells were used. PEI25k and unmodified polymer, poly(CBA-DAB) were also used as controls. All PEI25k-tretaed cells showed dramatically decreased cell viability depending on the concentrations, which indicates its severe cytotoxicity. It was found that poly(CBA-DAB) had little effect on the cell viability showing minimal cytotoxicity in all the cells examined due to its biodegradation in cytoplasm. In the case of poly(CBA-AG), the cytotoxicity was dependent on concentrations and cell types. Figure 5A shows that cytotoxicity of poly(CBA-AG) proportional to the concentration was observed in HeLa cells but it was lower than that of PEI25k. It may be associated with the growth inhibition and tumor cell death effect of agmatine residues in degraded fragments of poly(CBA-AG) to HeLa cells, which was already reported in other


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study although its mechanism is not fully understood.34 However, it was observed that poly(CBA-AG) exhibited no considerable cytotoxicity in Neuro 2a cells (Figure 5B) and A549 cells (Figure 5C), which was similar to or slightly lower than that of poly(CBA-DAB). Therefore, it was concluded that bioreducible poly(CBA-AG) exhibits low cytotoxicity.

Figure 5. MTT assay results in (A, D) HeLa cells, (B, E) Neuro 2a cells, and (C, F) A549 cells. (A-C) polymer results and (D-F) polyplex results.

The cytotoxicity of the polyplexes (pCN-Luci pDNA) was also examined (Figure 5D-F). Polymer concentration in polyplex (weight ratio=1) was set to be equivalent to polymer concentarion in polymer solution (1 µg/mL). PEI25k polyplexes showed significant cytotoxicity even at low concentrations in all cell lines, as expected. Cell viabilities of poly(CBA-DAB) polyplexes-treated cells were higher than 80% even at high concentrations (weight ratio of 100) in all cell lines, which means that cytotoxicity of poly(CBA-DAB) polyplexes is marginal. In the case of poly(CBA-AG) polyplexes, cells showed decreasing


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cell viability with the increase of concentration in HeLa cells, meaning agmatine moiety of polyplex also could induce cytotoxicity to HeLa cells in similar with the free polymer result (Figure 5D). In Neuro2A cells, poly(CBA-AG) polyplex-treated cells displayed high cell viability, which is similar with that of poly(CBA-DAB) polyplex-treated cells (Figure 5E). Although poly(CBA-AG) polyplex-treated cells showed 10-20% lower cell viabilities than poly(CBA-DAB) polyplex-treated cells in A549 cells, its cell viability was still over 80% even at high concentration (weight ratio of 80) in Figure 5F. Therefore, it was concluded that poly(CBA-AG) polyplex also shows low cytotoxicity.

3.6. Transfection experiments in vitro. To examine the gene transfection efficiency of poly(CBA-AG), luciferase transgene expression experiment was performed in HeLa, Neuro 2a, and A549 cells. PEI25k and poly(CBA-DAB) were used as controls. In the case of serumfree condition (Figure 6A, 6B, and 6C), poly(CBA-AG) showed highly enhanced transfection efficiency in comparison with unmodified backbone polymer, poly (CBA-DAB) in all cell lines at optimized weight ratios, which is similar to or even higher than that of PEI25k. This result was also observed in serum condition (Figure 6D, 6E, and 6F), meaning guanidine groups of poly(CBA-AG) could improve the transfection efficiency of the polymers as already reported in the previous work.18 However, when the weight ratio of the polyplexes increased, poly(CBA-AG) showed gradually decreased transfection efficiency in HeLa cells (Figure 6A and 6D). Transfection efficiency of polymers depends on various parameters such as cytotoxicity, DNA condensation and protection, serum stability, cellular uptake efficiency, or intracellular trafficking. In addition, an optimized weight ratio for the highest transfection efficiency is determined by the balance between the parameters. Therefore, disruption of the parameter balance based on increasing polymer amount may cause the decrease of transfection efficiency.


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On the other hand, it was found that the transfection efficiency of PEI25k was decreased significantly in serum condition in comparison with serum-free condition, which demonstrates that transfection of cationic polymers can be inhibited by nonspecific interaction with anionic serum proteins. However, in the case of both poly(CBA-AG) and poly(CBA-DAB), their transfection efficiencies in serum condition were similar to or slightly lower than those in serum-free condition in comparison with PEI25k result. This result indicates that poly(CBA-AG) polyplex possesses good serum stability and the potential to in vivo gene delivery system.

Figure 6. Transfection experiment results in (A,B, and C) serum-free condtion and (D, E, and F) serum condtion. The experiments were performed in (A,D) HeLa cells, (B,E) Neuro 2a, and (C,F) A549 cells, respectively. Numbers in small boxes mean the weight ratios of polyplexes. PEI25k polyplex was prepared at a weight ratio of 1.

3.7. Cellular uptake of polyplexes. The cellular uptake efficiency of poly(CBA-AG) polyplexes was measured by flow cytometry in HeLa and Neuro 2a cells in order to examine


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the effect of polyplex cellular uptake on the transfection efficiency. YOYO-1 Iodide-labeled pDNA was used for preparation of polyplexes. As shown in Figure 7, poly(CBA-AG) and poly(CBA-DAB) polyplexes showed increased cellular uptake efficiency with the increment of weight ratios and the cellular uptake efficiency of poly(CBA-AG) polyplexe was higher than that of poly(CBA-DAB) polyplex at all weight ratios. This result means that guanidine moiety of poly(CBA-AG) may enhance the cellular internalization of the polyplex. However, the cellular uptake efficiency didn’t show any significant correlation with transfection efficiency, considering transfection fficiency of PEI25k or those of poly(CBA-AG) at low weight ratios. Therefore, it is thought that the high transfection efficiency of poly(CBA-AG) may be induced by other factors such as nuclear localization ability of guanidine moieties as reported already in the previous study.18

Figure 7. Flow cytometry results of polyplexes in (A) HeLa and (B) Neuro 2a cells. (a) PEI25k: weight ratio=1, (b) poly(CBA-DAB): weight ratio=10, (c) poly(CBA-DAB): weight ratio=30, (d) poly(CBA-DAB): weight ratio=50, (e) poly(CBA-AG): weight ratio=10, (f)


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poly(CBA-AG): weight ratio=30, (g) poly(CBA-AG): weight ratio=50. % values are cellular uptake efficiencies of polyplexes (YOYO-1 Iodide-labeled pDNA) by setting the divided regions as gates.

3.8. Intracellular trafficking of polyplexes. We observed the intracellular trafficking of polyplexes by confocal microscopy after 4 h of polyplex treatment. The nuclei of HeLa cells were stained by Hoechst 33342 (blue) and acidic compartments such as late-endosome or lysosome were stained by LysoTracker red (red). pDNA was labeled by YOYO-1 Iodide (green) for observing the location of the delivered pDNA. As shown in Figure 8, pDNA of PEI25k polyplex showed particle-like fluorescence in cells and they almost seemed to be located in cytosol, not nuclei of the cells. In similar with PEI25k, green dot-like pDNA fluorescence of poly(CBA-DAB) polyplex was also observed to be mainly present in cytosol. This result means that pDNA delivered by PEI25k or poly(CBA-DAB) couldn’t reach the cell nuclei at this time scale and reduction-sensitive degradation of poly(CBA-DAB) polyplex was not sufficient to dissociate pDNA from the polyplex in this cytosol condition, although it is a bioreducible polymer.


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Figure 8. Intracellular trafficking observation by confocal microscopy in HeLa cells. The nuclei were stained by Hoechst 33342 (blue) and acidic compartments such as late-endosome or lysosome were stained by LysoTracker red (red). pDNA was labeled by YOYO-1 Iodide (green).

However, in the case of poly(CBA-AG), it was observed that strong and well distributed pDNA fluorescence was intensively located in cell nuclei. Taken together with degradation result of poly(CBA-AG) and the above confocal microscopy observation for poly(CBADAB), it is thought that self-catalyzed hydrolysis of guanidine moieties and reductive cleavage of internal disulfide bonds in cytosol can facilitate the pDNA dissociation from the polyplexes cooperatively. This result means that poly(CBA-AG) can deliver pDNA efficiently into cell nucleus and it may be due to the efficient release of pDNA from the polyplexes by intracellular biodegradation of the polymer and to the nuclear localization ability of guanidine moieties, which are previously suggested.18

3.9. Anticancer activity by apoptin gene delivery by poly(CBA-AG). Antitumor effect of poly(CBA-AG) polyplexes via apoptotic gene delivery was assessed by MTT assay in A549 and Neuro 2a cells. pJDK-apoptin pDNA was used as a apoptic gene, encoding the expression of s apoptin protein which is an antitumor agent inducing tumor cell-selective apoptosis.26 PEI25k and poly(CBA-DAB) polyplexes were used as controls. The apoptotic activity of expressed apoptin was indirectly judged by measuring relative cell viability after treatment of the polyplexes. In Figure 9A, poly(CBA-AG) polyplex exhibited a powerful cytotoxic effect on A549 cells in comparison with poly(CBA-DAB) polyplex. It showed more than 80% decrease of cell


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viability at a weight ratio of 70 and it was much more effective than that of PEI25k polyplex (50% decrease). Similarly to A549 cell result, cancer cell-killing effect of poly(CBA-AG) polyplex via apoptin gene delivery was found to be higher than that of poly(CBA-DAB) or PEI25k polyplex in Neuro 2a cells (Figure 9B). Considering the minimal cytotoxicity result of poly(CBA-AG) and its polyplex by MTT assay in A549 and Neuro 2a cells, this result demonstrates the potential of poly(CBA-AG) for cancer gene therapy via apoptin gene delivery. RT-PCR analysis was performed in order to identify the apoptin gene expression in A549 cells (Figure 9C). GAPDH house keeping gene was used as a control. All apoptin cDNA bands were observed from PCR products of polyplexes (pJDK-apoptin)-treated cells, except untreated cell control, meaning apoptin was successfully expressed in cells after transfection.


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Figure 9. Anticancer activity evaluation by MTT assay in (A) A549 cells and (B) Neuro 2a cells. Numbers in small boxes mean the weight ratios of polyplexes. (C) RT-PCR result. DAB70: poly(CBA-DAB) weight ratio=70, AG30: poly(CBA-AG) weight ratio=30, AG50: poly(CBA-AG) weight ratio=50, AG70: poly(CBA-AG) weight ratio=70.

Annexin V/PI apoptosis assay was also performed in order to identify the anticancer activity by apoptosis mediated by apoptin gene delivery in A549 cells. As shown in Figure 10, poly(CBA-DAB) just exhibited about 26% of apoptosis even at a weight ratio of 100. PEI25k showed about 69% of apoptosis. However, poly(CBA-AG) showed about 80% of apoptosis at a weight ratio of 50 and about 90% of apoptosis at a weight ratio of 100. This result is consistent well with the previous MTT assay and demonstrates that apoptin gene delivery by poly(CBA-AG) could induce high degree of apoptosis to cancer cells.

Figure 10. Anticancer activity evaluation by Annexin V/PI apoptosis assay in A549 cells. (A) cell only, (B) PEI25k: weight ratio=1, (C) poly(CBA-DAB): weight ratio=100, (D) poly(CBA-AG): weight ratio=30, (E) poly(CBA-AG): weight ratio=50, (F) poly(CBA-AG): weight ratio=100. Numbers in boxes mean % cell population value of each divided region.

4. Conclusions


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Endogenous neurotransmitter, agmatine moiety was introduced to synthesize bioreducible polymer, poly(CBA-AG) through Michael addition of CBA and N-Boc-DAB followed by guanidinylation for gene delivery systems. poly(CBA-AG) could form nano-sized and positively charged polylexes with pDNA, which can be degraded in reducing environment due to reducible cleavage of internal disulfide bonds. Moreover, it can be degraded in normal aqueous condition unlike backbone polymer, poly(CBA-DAB) and it is probably due to selfcatalyzed hydrolysis of internal amide bonds by guanidine moieties. poly(CBA-AG) showed cell-dependent but marginal cytotoxicity and also showed highly enhanced transfection efficiency in comparison with poly(CBA-DAB). Cellular uptake efficiency of the polyplexes is not considered to contribute its high transfection efficiency significantly but pDNA localization ability of guanidine moieties and self-catalyzed degradation by guanidine moieties as well as disulfide bonds cleavage in cytosol are thought as important factors to improve the transfection efficiency. It was also confirmed that poly(CBA-AG)/pJDK-apoptin polyplex showed higher anticancer activity mediated by apoptosis than PEI25k or poly(CBADAB) polyplex. In conclusion, poly(CBA-AG) possesses a potential for non-toxic and efficient gene delivery systems and this work about bioreducible polymer with dual biodegradability would be a guide for further research about development of biodegradable polymers.

Supporting Information Available: degradation analysis of poly(CBA-AG) and poly(CBADAB), characterization of poly(CBA-DAB) polyplex. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments


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This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0015045) and by the Ministry of Science, ICT, and Future Planning (NRF-2014R1A1A1037692). We also acknowledge the National Instrumentation Center for Environmental Management (NICEM) for permission to take

1

H NMR and CLSM

observation, Prof. Cheol-Heui Yun for permission to take flow-cytometry measurements, and Prof. Yan Lee for permission to take Zeta-sizer measurements.


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Biomacromolecules

TOC Agmatine-containing bioreducible polymer for gene delivery systems and its dual degradation behavior


Agmatine-Containing Bioreducible Polymer for Gene Delivery

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