Redox-Responsive Hyperbranched Poly(amido amine)s with Tertiary

May 21, 2013 - All the PAAs were able to condense pDNA into nanoparticles with diameters ... available by participants in Crossref's Cited-by Linking ...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/Biomac

Redox-Responsive Hyperbranched Poly(amido amine)s with Tertiary Amino Cores for Gene Delivery Yuan Ping,† Decheng Wu,‡ Jatin Nitin Kumar,† Weiren Cheng,† Chee Leng Lay,† and Ye Liu*,† †

Institute of Materials Research and Engineering, A*STAR (Agency for Science, Technology and Research), 3 Research Link, Singapore 117602, Singapore ‡ Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: Redox-responsive hyperbranched poly(amido amine)s (PAAs) with tertiary amino cores and amine, poly(ethylene glycol) (PEG) and hydroxyl terminal groups were prepared for DNA delivery respectively. The DNA condensation capability of PAAs was investigated using gel electrophoresis, and the results showed that PAA terminated with 1-(2-aminoethyl)piperazine (AEPZ) (BAA) is the most efficient in binding plasmid DNA (pDNA). The diameter and zeta-potential of polyplexes from PAAs were characterized using dynamic light scattering (DLS), and the morphology of the polyplexes was obtained using atomic force microscopy (AFM). All the PAAs were able to condense pDNA into nanoparticles with diameters between 50 and 200 nm with a positive surface charge when the weight ratio of polymer/DNA was higher than 20. Glutathione (GSH)-induced DNA release from polyplexes and the buffering capability of PAAs were investigated as well. Cytotoxicity of PAAs and in vitro gene transfection of polyplexes were evaluated in HEK293, COS-7, MCF-7 and Hep G2 cell lines, respectively. The results reflect that PAAs show remarkably low or even no cytotoxicity, and that PAA with amino terminal groups mediates the most efficient gene transfection with the transfection efficiency comparable to that of 25 kDa polyethylenimine. Further the effects of the presence of buthionine sulfoximine (BSO) on the transfection efficiency and cytotoxicity of BAA polyplexes were investigated.

■

research groups for gene delivery.18,20−25 There are three key factors affecting the transfection efficiency of PAAs. The first factor is the topological structure of PAAs. Perfect dendritic PAAs show lower transfection efficiency than its degraded derivatives with a lower branching degree, however, polyplexes from branched PAAs were generally found to yield higher transfection efficiencies than those from their linear analogs. The most important feature of hyperbranched PAAs is that their transfection activity could be further enhanced by the presence of serum partially due to the reduced cytotoxicity induced by protein complexation. Thus, this unique property of hyperbranched PAAs which leads to a transfection efficiency higher than that of the “gold standard” polyethyleneimine (PEI) of 25 kDa will become very promising for in vivo application.22 Second, various chemical functionalities in cationic polymers profoundly influence the gene transfection

INTRODUCTION Safe and efficient delivery of exogenous deoxyribonucleic acid (DNA) into host cells is one of the major challenges to the clinical success of gene therapy. Although so far viral vectors are most frequently used to deliver large size genetic materials, the problems related to their intrinsic safety, complexity and undesired side effects are still daunting hurdles to clinical applications.1 In comparison, nonviral vectors can be prepared from materials with controllable structures without host immunogenicity.2−4 However, the relatively poor transfection efficiency of nonviral vectors due to the difficulties in overcoming the extra- and intracellular delivery barriers has severely limited their successful applications in clinical trials. Among various types of polymer systems explored for gene delivery,5−14 poly(amido amine)s (PAAs) have been shown to be a versatile class of polymers promising for gene delivery due to their properties including biodegradability, biocompatibility, generally low hemolytic activity and peptide-mimicking properties.15−19 In particular, disulfide-containing bioreducible linear or branched PAAs have been favorably exploited by several © XXXX American Chemical Society

Received: April 2, 2013 Revised: May 9, 2013

A

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

■

activity.25 The effects of terminal groups on transfection efficiency have been investigated by Engbersen and co-workers recently. Several bioreducible hyperbranched PAAs were synthesized and their linear analogues, which were end-capped with either 4-aminobutanol (ABOL) or with 2-aminoethanol (ETA) were investigated for gene transfection.22 However, this study only employed alcohol-containing molecules to cap the PAA terminal. Thus, it would be desirable to know the effect of using other types of capping groups on the transfection efficiency of PAAs. Third, the introducing of bioreducible disulfide linkages in PAA backbone not only facilitates the rapid unpacking of DNA in cytoplasm and nucleus where glutathione levels are thousand-fold higher than in the extracellular environment to achieve high transfection efficiency, it can also help to reduce cytotoxicity by avoiding accumulation of high-molecular-weight cationic polymers inside the cells.26 Branched PAAs prepared via Michael addition polymerization of tetrafunctional amino compounds and bisacrylamides were accompanied by gel formation.22 Also, branched PAAs were prepared via Michael addition polymerization of trifunctional amino compounds with three types of amines of different reactivity. Branched PAAs containing secondary amines and tertiary amines in the cores can be prepared via polymerization of trifunctional amino compounds with equimolar bisacrylamides,18 and Chen et al. also reported that the preparation of hyperbranched PAAs containing secondary amines and tertiary amines in the cores could be obtained via Michael addition polymerization of a linear aliphatic trifunctional amino compound with double molar bisacrylamide.24 In contrast, our previous works have shown that Michael addition polymerization of cyclic aliphatic trifunctional amino compounds with double molar bisacrylic monomers, i.e., 2A2 + BB′B″ approach,14,27,28 could produce hyperbranched polymers with only tertiary amines in the cores and well-defined hyperbranched structures.29 It should be noted that tertiary amine groups in the polymer backbone play an important role in gene transfection. For example, poly((2-dimethylamino)ethyl methacrylate), which contains only tertiary amines, can mediated 90% of the transfection efficiency of branched PEI (25 kDa). The tertiary amines in PDMAEMA is not only responsible for DNA complexing, but also provide strong endosomolytic activity due to its proton buffering effect.30,31 In the case of PAA, the tertiary amine groups in the linear PAAs can be protonated, which renders the polymer cationic property and generally a good solubility in water. It was also reported that PAAs are subjected to conspicuous conformational changes upon the protonation of tertiary amines, which endows themselves endosomolytic capability that can facilitate polyplexes to access cytoplasm after endosomal escape.32 Considering the important effects of the topological structures and chemical composition on the gene transfection efficiency, we are motivated to investigate the performance of hyperbranched PAAs with tertiary amino cores for gene delivery. Here, a series of hyperbranched bioreducible PAAs of tertiary amino cores with different terminal groups were developed. Their biophysical properties and structure−activity relationships in transfection efficiency were investigated, and the reduction-sensitivity of the PAA with the highest transfection efficiency and serum-enhanced transfection efficiency properties were studied.

Article

EXPERIMENTAL SECTION

Materials and Methods. Materials. 1-(2-aminoethyl)piperazine (AEPZ), N,N′-bis(acryloyl)cystamine (BAC), 3-amino-1,2-propanediol (APD), branched PEI with average molecular weight of 25 kDa, 1(4,5-dimethyl-thiazol-2-yl)-3,5-diphenylfor-mazan (MTT), L-buthionine sulfoximine (BSO) were purchased from Sigma-Aldrich and used as received. All solvents used in this study were purchased from Tedia. α-Amino-ω-methoxy-poly(ethylene glycol) with either molecular weight of 350 (PEG350) or 750 (PEG750) was prepared from poly(ethylene glycol) methyl ether according to the reported literature.33 Plasmid pRL-CMV of 4.1 kb size encoding Renilla luciferase driven by CMV promoter was purchased from Promega Corporation (Madison, USA). Plasmid pEGFP-N1 encoding a red-shifted variant of wild-type green fluorescence protein (GFP) was purchased from Clontech Laboratories Inc. (Palo Alto, USA). All plasmids were amplified in Escherichia coli and purified with Endofree Mega plasmid purification kit supplied by Qiagen (Hilden, Germany). The purified plasmid DNA was dissolved in Tris-EDTA (TE) buffer (10 mM TirsHCl, 1 mM EDTA, pH = 7.5), and its concentration and purity was tested by NanoDrop 2000 spectrophotometer (Wilmington, USA). HEK293 (human embryonic kidney cell line), COS-7 (African green monkey kidney fibroblast cells line), Hep G2 (human hepatoma cell line), and MCF-7 (human breast adenocarcinoma cell line) were obtained from American Type Culture Collection (ATCC, Rockville, MD). They were maintained in Dulbecco’s modified eagle medium (DMEM, invitrogen) with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml penicillin and 100 μg/mL streptomycin at 37 °C in an incubator with 5% CO2 atmosphere. Synthesis of PAA Polymers. The bioreducible poly(BAC2-AEPZ1)AEPZ polymer was synthesized by one-pot two-step Michael addition polymerization as an example according to our previous methods with a slight modification.29 In brief, BAC (3.0 mmol) was first dissolved in 10 mL of methanol at room temperature. AEPZ (1.5 mmol) was then added dropwise to the solution while stirring, followed by rinsing with 2 mL of methanol. The mixture was stirred at 50 °C for 6 days. After that, 3.2 mmol of AEPZ was added and was kept stirring at 60 °C for 3 days to seal terminal vinyl groups. The resulting mixture was added to excessive diethyl ether under vigorously stirring. The precipitate was isolated by centrifugation, dissolved in methanol, and precipitated in 100 mL of acetone containing 5 mL of 37% concentration HCl. The final product isolated from acidified acetone was dried under vacuum at 50 °C for 24 h. For the synthesis of other PAA polymers, all the synthesis procedures were the same as those of BAA except that APD, AEPZ/PEG350 mixture (molar ratio: 1:1), PEG350, or PEG750 was used to seal terminal vinyl groups. Molecular weight was determined from gel permeation chromatography (GPC) according to our previous reported method.29 Characterize of PAA Polymers. The structure of PAAs was characterized by carbon-13 nuclear magnetic resonance spectroscopy (13C NMR). The 13C NMR spectra were recorded on a Bruker AV400 NMR spectrometer at 100 MHz at room temperature. The 13C NMR measurements were carried out using composite pulse decoupling with an acquisition time of 0.82 s, a pulse repetition time of 5.0 s, a 30° pulse width, 20 080-Hz spectral width, and 32 K data points. All the spectra were evaluated with the NMR data processing software Topspin 1.3. Preparation of the Polymer/DNA Complexes. The plasmid DNA (pDNA) stock solution was diluted to the desired concentration with TE buffer (pH 7.5) before use. Polymers dissolved in TE buffer (pH 7.5) were added dropwise to DNA solution in equivalent volume according to the predetermined weight ratios. The mixture solution was vortexed and incubated for 30 min at room temperature and for the subsequent experiments. The polyplexes were always freshly prepared before each experiment. Gel Electrophoresis Assay. The electrophoretic mobility of the polymer/DNA polyplexes was examined for their ability to inhibit pDNA migration through gel electrophoresis experiment. In general, 20 μL of polyplexes solution containing 0.4 μg was withdrawn to mix B

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

with 5 μL of 5× loading buffer, and the resulted solution mixture was analyzed on 0.9% agarose gel containing 1× thiazole orange. Gel electrophoresis was carried out in 1× Tris-acetate-EDTA (TAE) buffer (40 mM Tris-acetate, 1 mM EDTA, pH 7.5) at 100 V for 45 min in a Sub-Cell system (Bio-Rad Laboratories, CA). DNA bands were visualized by a LAS-4000 luminescent image analyzer (Fujifilm, Japan). The ability of polymer to complex pDNA was also evaluated using a dye-exclusion technique with PicoGreen. Briefly, 50 μL of polyplexes were mixed with diluted PicoGreen dsDNA reagent (Invitrogen) according to the manufacturer’s instruction. After incubation for 10 min in dark, the fluorescence intensity was then measured on an Infinite M200 microplate reader (Tecan, Switzerland) with standard fluorescein wavelengths (excitation 480 nm, emission 520 nm). The relative fluorescence was calculated as fluorescence induced by polyplexes against the fluorescence induced by DNA alone (without polymer). The fluorescence of PicoGreen was used as the blank. Dynamic Light Scattering (DLS) Study. The hydrodynamic size and zeta potential of polyplexes were measured on a Zetasizer Nano ZS (Malvern Instruments Ltd., MA, USA), with a laser light wavelength of 633 nm at a 173° scattering angle. One hundred microliters of polyplex containing 3 μg of pDNA was prepared at various weight ratios. The mixture was vortexed and incubated for 30 min at room temperature before being diluted to 900 μL by 15 mM of KCl solution at the time of measurement. For zeta potential measurements, polyplexes were formed at various weight ratios in 0.1 M of KCl solution and were adjust to the desired pH (pH 7.5) in a similar way as above. The measurements were conducted in a capillary zeta potential cell in automatic mode. Atomic Force Microscopy (AFM). The polyplex morphology was further visualized by an AFM system on a Dimension 3100 model with a Nanoscope IIIa controller (Veeco, Santa Barbara, USA) according to the reported method.34 Two microliters of BAA complex containing 0.01 μg of pDNA at a weight ratio of 45 was dropped onto on mica sheets and dried in a cabinet drier overnight. The samples were imaged using the tapping mode with setting of 512 pixels/line and 1 Hz scan rate. Image analysis was performed using Nanoscope software after removing the background slope by flatting images. To evaluate the reduction sensitivity of BAA, the appropriate amount of 0.1 M dithiothreitol (DTT) was added to the polyplex solution to achieve the final DTT concentration of 10 μM or 10 mM. The solutions were incubated on Eppendorf Thermomixer at 37 °C with constant shaking for 2 h before above measurements. Buffering Capacity. The buffering capacity of PAA polymers over the pH range from 10 to 4 as determined by acid−base titration according to the previously reported method.31 Briefly, PAA polymer was dissolved in 10 mL of 0.15 M sodium chloride to obtain a 20 mM amino group concentration, which was subsequently adjusted by 0.1 M NaOH or HCl to an initial pH 10.0. Then, the basic polymer solution was titrated to around pH 4.0 with aliquots of 0.1 M HCl. pH values of the solutions were measured by a Metrohm 826 pH meter (Herisau, Switzerland). PEI of the same amino group concentration was used as controls, and 0.15 M sodium chloride was used as the blank solution. In Vitro DNA Release. To evaluate the DTT-induced release of pDNA from BAA polymer in vitro, 100 μL of BAA/DNA complexes prepared in TE buffer (10 mM Tirs-HCl, 1 mM EDTA, pH = 7.5) were incubated with or without DTT. The polyplexes in microcentrifuge tubes were shaken on an Eppendorf Thermomixer at 37 °C at different time scales, and all the polyplexes were stored at 4 °C until fluorescence measurement. Fifty microliters of polyplexes was further mixed with diluted PicoGreen dsDNA reagent (Invitrogen) according to the manufacturer’s instruction. After incubation for 10 min in a black 96-well plate, the fluorescence intensity was then measured on a Infinite M200 microplate reader (Tecan, Switzerland) with standard fluorescein wavelength (excitation 480 nm, emission 520 nm). The release DNA amount was calculated from the standard curve calibrated with DNA samples of known concentration. The percentage of pDNA released from BAA was calculated by using the following equation: Cumulative DNA release (%) = [(DNAR − DNAN)]/[(DNAT − DNAN)] × 100%, where DNAR is the released DNA amount, DNAT is the total DNA amount, and DNAN is nonbound DNA amount.

In Vitro Transfection and Luciferase Assay. For in vitro transfection studies using plasmid pRL-CMV, cells were seeded onto 24-well plate at a density of 5 × 104 per well in 0.5 mL of culture medium 24 h prior to transfection. At the time of transfection, the medium in each well was replaced with either 0.3 mL of Opti-MEM reduced serum medium (Invitrogen) or normal DMEM. Twenty microliters of polyplexes containing 1 μg plasmid DNA was added to each well and was incubated with the cells for 4 h under standard culture condition. Then the medium was replaced with 0.5 mL of fresh culture medium and cells were further incubated for 20 h under the same conditions, resulting in a total transfection time of 24 h. At the end of transfection, cells were washed with preheated phosphate buffered saline (PBS; pH 7.4) twice, and lysed with 100 μL of cell lysis buffer (Promega, USA) for 30 min. The luciferase activity in cell extracts was measured using a luciferase assay kit (Promega, USA) on a single-well luminometer (Berthold lumat LB9507, Germany) for 10 s. The relative light units (RLUs) were normalized against protein concentration in the cell extracts, which was measured using a bicinchoninic acid assay kit (Biorad, CA, USA). Absorption was measured on a microplate reader (SpectraMax Plus384, Molecular Devices) at 570 nm and compared to a standard curve calibrated with BSA samples of known concentration. Results are expressed as relative light units per milligram of cell protein lysate (RLU/mg protein). For DNA transfection mediated by BAA in the presence of BSO, cells were first incubated with 0.2 mM BSO for 72 h before GSH level was detected by Glutathione Assay Kit according to the manufacturer’s instruction. After verification of GSH level, transfection was carried out with the same protocol except that the culture medium was maintained at the BSO concentration of 0.2 M until the end of cell transfection. Cytotoxicity of PAA Polymers and Polyplexes. Four cell lines (HEK293, COS-7, MCF-7 and Hep G2) were cultured in culture medium supplemented with 10% FBS at 37 °C, 5% CO2, and 95% relative humidity. 100 μL of cells were seeded into 96-well plates (NUNC, Wiesbaden, Germany) at a density of 1 × 105 cells/mL. After 24 h, culture media were replaced with serum-rich culture media containing either serial dilutions of polymers or polyplexes of different polymer/DNA weight ratio. The serial dilutions of polymers were further incubated with cells for 24 h, whereas polyplexes of different polymer/DNA weight ratio were first incubated with cells for 4 h and then the medium was replaced with 0.1 mL of fresh culture medium before cells were further incubated for 20 h under the same conditions. Subsequently, 10 μL of 5 mg/mL filtered MTT stock solution was added to each well, and unreacted dye was removed by aspiration after 4 h. The formazan crystals were dissolved in 100 μL/well DMSO and measured in a microplate reader (SpectraMax Plus384, Molecular Devices) at a wavelength of 570 nm. Confocal Laser Scanning Microscope Observation. pDNA was first labeled with CX-Rhodamine using the Label IT Nucleic Acid Labeling Kit (Mirus, Madison, WI) according to the manufacture’s protocol. Hep G2 cells were seeded on a 35-mm glass base dish (Iwaki, Japan) and incubated in 1.5 mL of DMEM supplemented with 10% FBS . After 24 h, the medium was replaced with fresh medium, in which 60 μL of BAA polyplex solution containing 2 μg of rhodaminelabeled pEGFP-N1 plasmid were added. After transfection, the cells were washed twice with prewarmed PBS (pH 7.4), and were further fixed with 4% paraformaldehyde for 20 min. Cell nuclei were stained with ProLong Gold Antifade Reagent with DAPI (Invitrogen, USA) after the removal of paraformaldehyde residues. The cells were imaged under a confocal laser scanning microscope (FV1000, Olympus, Japan) with a 60 × objective to visualize the fluorochromes with the following excitation (Ex) and emission (Em) wavelength: DAPI (Ex: 350 nm, Em: 470 nm), GFP (Ex: 488 nm, Em: 515 nm) and CXRhodamine (Ex: 572 nm, Em: 590 nm). Flow Cytometric Analysis of Cellular Uptake and GFP Expression. Hep G2 cells were seeded on a 12-well plate at the cell density of 20,000 cells/well in DMEM supplemented with 10% FBS 24 h prior to transfection. At the time of transfection, the medium in each well was replaced with serum-rich medium, and 20 μL of polyplexes containing 2 μg rhodamine-labeled pEGFP-N1 plasmid were added to each well. After different time of incubation, cells were washed three times with C

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Scheme 1. Scheme of Synthesis of Various Reduction-Sensitive PAAs

PBS (pH 7.4), detached from plate with trypsin, and finally suspended in PBS solution (pH 7.4). The fluorescence intensity of rhodaminelabeled pDNA and the percentage of positive GFP cells in the suspended solutions were measured using a BD LSR II Flow Cytometer (BD Biosciences).

■

react with the precursor as typical amine terminated PAAs. Poly(ethylene glycol) (PEG)-terminated PAAs were prepared because of the well-established property of PEG such as avoiding nonspecific adsorption of proteins, and α-amino-ωmethoxy-PEG with molecular weights of 350 (PEG350) and 750 (PEG750) were used. Also a mixture of AEPZ and PEG350 (1:1) was used. Meanwhile, APD-terminated PAAs were prepared as typical alcohol-terminated PAAs. The 13C NMR spectra of nonterminated PAA precursor (BA), BAA, BAAP, BAP1A1, BAP350 and BAP750 are shown in Figure 1 and Figure S1. While the typical carbon signals attributed to the vinyl groups of acrylate moiety were well observed in δ123− 133 ppm in BA spectrum, the disappearance of these peaks was found for all PAA polymers, indicating complete end-capping via Michael addition reaction. All of the characteristic peaks of PAA polymers can be well identified from the spectrum. For example, as compared with BA spectrum, the characteristic signals appeared in δ69.5−63.4 ppm, which correspond to the APD carbons, and these signals were clearly differentiated from those carbon signals from BA core. In the case of BAP1A1, BAP350, and BAP750, the major carbon peaks from the

RESULTS AND DISCUSSION

Synthesis and Characterization of Hyperbranched PAAs with Tertiary Amino Cores with Different Terminal Groups. The hyperbranched PAAs with tertiary amino cores were synthesized via the 2A2+BB′B″ approach according to our previous method.29 As shown in Scheme 1, the precursor, BA, with terminal vinyl groups (PAA core) was first produced by the reaction of a AEPZ and a double-molar BAC. Three typical types of terminal species were chosen as the ending caps in an attempt to seal the vinyl groups of PAA cores. Our previous work showed that no significant difference in DNA transfection efficiency was observed for hyperbranched poly(amino ester)s with primary amine, secondary amines and tertiary amine as terminal groups, respectively.14,27 Based on these studies, we prepared primary amine terminated PAAs via using AEPZ to D

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 1. 13C NMR spectrum of BA in CD3OD from δ75−25, and BAA, BAAP, BAP1A1, BAP350, and BAP750 from δ75−25 ppm in D2O.

repeating unit of PEG were δ69.6 ppm (BAP350 and BAP750) and δ70.3 ppm (BAP1A1). All the evidence strongly indicates the successful end-capping with our selected terminal capping species. The molecular weight of PAAs and polydispersity index (PDI) are summarized in Table S1, and GPC traces are also reported in Figure S2. On the basis of the GPC analysis results, the repeating unit of the PAA core was estimated to be 22.8, which is in agreement with the static light scattering results (19.4). Biophysical Characterization of PAA Polyplexes. Polycations can spontaneously interact with DNA to form polyplexes through electrostatic interactions. In this work, the ability of PAAs to condense plasmid DNA into particulate structures was examined by agarose gel electrophoresis, particle size, and zeta potential assays. As shown in Figure 2, both PEGterminated PAAs required higher polymer/DNA weight ratios to completely inhibit DNA migration. BAP350 required a weight ratio of 8 to inhibit pDNA mobility and BAP750 required a weight ratio of 14 to completely inhibition pDNA mobility. Due to the steric hindrance of the PEG chain as well as the high degree of PEGylation, the large amounts of cationic polymers are believed to be necessary in achieving complete DNA complexation.35 In addition, as the length of PEG chain may also affect the formation of the polyplexes, it seems reasonable for BAP750 to exhibit poorer complexation capability than that of BAP350.36 In the case of BAP1A1 where the terminal ratio of PEG350 to AEPZ was close to 1, pDNA complexation capability was significantly improved and the full complexation could be achieved at the weight ratio of 4 or below. BAP terminated with AEPZ, which could retard DNA strongly within the weight ratio of 4, was observed to be more efficient in DNA retardation in comparison with BAAP that was terminated by APD. The weaker DNA complexation ability of

BAAP relative to BAA was probably due to the absence of primary amines, which are believed to be important in DNA condensation.37 When the BAA polyplexes were exposed to 10 mM DTT, rapid DNA decomplexation occurred, and released free DNA bands were clearly visible at all tested weight ratios, which was due to the degradation of BAA. These results together indicate that terminal groups of PAA in different structures can significantly affect the interaction between PAA and pDNA and that PAA fully or partially terminated with AEPZ bearing trifunctional amines is able to render stronger pDNA complexation in comparison with PAA terminated with either PEG or alcohol-containing molecules. The ability of PAA polymers to complex pDNA was also assessed by using Picogreen agent, which is an ultrasensitive fluorescent nucleic acid stain quantitating double-stranded DNA (dsDNA) in solution. As displayed in Figure 2B, the fluorescence intensity of all PAA/DNA complexes decreased progressively with increasing weight ratios, suggesting uncomplexed free DNA reduced when increasing the polymer concentration. BAP1A1 and BAAP showed a very similar trend in terms of DNA complexation ability, and both of them reached a relative fluorescence plateau around 20% at the weight ratio 6 and above, suggesting the relatively complete DNA complexation. BAA showed stronger DNA complexation ability as compared to BAAP or BAP1A1, and it was found that complete DNA complexation can be reached at a weight ratio of 4. On the other hand, PEGylated PAA polymers showed relatively poor complexation ability as compared with other PAAs due to the steric hindrance of PEG as discussed earlier. Whereas BAP350 showed complete complexation at weight ratio of 10, BAP750 was not able to fully complex pDNA even at the weight rato of 14. When DTT was added BAA polyplex solution, decomplexation was detected, and relative fluoresE

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 3. DLS study of (A) particle size and (B) zeta potential of polyplexes formed by plasmid DNA (pRL-Luc) and PAA polymers at various weight ratios.

Figure 2. (A) Electrophoretic mobility of pDNA in the polyplexes formed by hyperbranched PAA polymers at different polymer/DNA weight ratios. The pDNA mobility in BAA polyplexes was also tested in the presence of 10 mM DTT. pDNA without polymer complexation was used as control. (B) PicoGreen dye exclusion assay of polymer/ DNA complexes at weight ratios from 0 to 14.

of DNA effectively, it should also render polyplexes with a positive surface charge that can electrostatically interact with negatively charged cellular membranes. As indicated in Figure 3B, the surface charge of PAAs, both fully (BAP350 and BAP750) and partially (BAP1A1) terminated with PEG showed similar levels and trends of zeta potential with the increased weight ratio, ranging from 10 to 30 mV. These PEGterminated PAAs always displayed lower surface charge as compared to that of BAA or BAAP at the same ratio, possibly due to the shielding effect of neutral PEG on the polyplex surface. In the case of BAA polyplexes, the zeta potential increases sharply from 7.4 to 24.1 mV with a weight ratio increase from 10 to 90, reaching a plateau around 40 mV at a weight ratio of 40. The morphology of BAA polyplex and the unpacking of DNA from BAA polyplexes were visualized using the AFM. Representative taping mode AFM images of BAA/DNA at the weight ratio of 60 are shown in Figure 4. Naked DNA was displayed as supercoiled structures, whereas BAA/pDNA complexes were observed to be compacted spherical particles with the diameter range of 50−200 nm. In the presence of 10 mM DTT, the BAA/pDNA complex became unstable. Larger particles with loose and irregular morphology were detected, suggesting the degradation of BAA was initiated by DTT. The result is consistent with that obtained from particle size measurement through DLS (Figure 4B). The above results indicate that the complexation of pDNA by the PAA led to the formation of dense nanoparticles that are small enough to be readily endocytosed by cells. After the escape from endosome, the bioreducible PAA backbone would facilitate the rapid unpacking of DNA in cytoplasm and nucleus under the effect of glutathione to achieve high transfection efficiency.

cence almost remained unchanged in the range of 90−100%. All these results are in agreement with the gel retardation assays. A successful gene delivery system requires that DNA must be condensed by polycation into polyplexes small enough to facilitate cellular uptake.38 Typically, positively charged polyplexes with sizes smaller than 200 nm are not only endocytosed into the cells more readily through clathrin-coated pit mechanisms for in vitro transfection, they are also favorable for in vivo distribution.39 To investigate whether hyperbranched PAAs were able to condense DNA efficiently, particle size was first studied as a function of weight ratio through DLS analysis. As presented in Figure 3A, particle size of all PAA polyplexes generally decreased with the increased weight ratio. Except for BAAP750, all PAA polyplexes could form nanoparticles within 200 nm at the weight ratio of 10 or higher. BAA yielded smaller polyplexes than other polymers at low polymer/ DNA weight ratios. All the polyplexes were found to be stabilized in the range of 75−110 nm at weight ratio of 45 or above and further increase of the weight ratio did not significantly change the particle size of these polyplexes. These results indicate that polymer/pDNA complexes within this size range can readily undergo endocytosis. Zeta potential is an indicator of the surface charge on the polymer/pDNA nanoparticles. For charge-mediated gene transfer, polymer should not only possess the ability to shield the anionic charge F

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

BAA polyplex nanostructure remained relatively stable. Hence, this fact implies the biodegradable nature of BAA may greatly facilitate the pDNA release as a consequence of thiol-induced degradation of polyplexes through the cleavage of disulfide bonds, and in turn, modulate the gene expression in vitro or in vivo. Efficient escape from endosomes is one of the most important factors to be considered for the design of gene delivery vehicles. This event is associated with the buffering capacity of gene vectors within the pH range of 7.4 to 5.1, in which vectors undergo shift from neutral extracellular environment to the slightly acidic environment of the endosome. By disrupting the endosomal membrane, polycations with high buffering capacity is able to mediate efficient escape from endosome to cytosol triggered by the “proton-sponge” effect. In this study, acid−base titration was performed to evaluate the proton-buffering effect of PAA polymers. As depicted in Figure 6, titration of 0.15 M sodium chloride solution resulted in a

Figure 4. (A) AFM images of naked pDNA, polyplexes (formed by BAA and pDNA at polymer/DNA weight ratio of 60), and DTTinduced polyplexes in amplitude mode. The bar represents 500 nm. (B) Particle size change of BAA in the presence or absence of DTT measured by DLS.

The DNA release from the degradable polymer matrix may have a significant effect on gene transfection efficiency. We hypothesized that the intracellular GSH level would influence the release kinetics of pDNA and, as a result, may lead to a rapid gene expression upon the degradation of the PAA. In order to understand DNA release kinetics in the presence of DTT, we further quantified the DNA release percentage at various time points through the use of Picogreen agent, which is an ultrasensitive fluorescent nucleic acid stain quantitating double-stranded DNA (dsDNA) in solution. As presented in Figure 5, BAA polyplexes could barely release DNA from its

Figure 6. Acid−base titration curves for PAA polymers, PEI (25 kDa) and blank (0.15 M NaCl) solution from pH 10 to 3.

nearly vertical curve, indicating its neglectable buffering capacity. Titration of BAA and BAP1A1 solutions showed a similar trend of pH change, and there was no significant difference in buffering capacity between them within the pH range of 7.4 to 5.0. In case of BAAP, BAP350 and BAP750, they exhibited a similar trend but with lower buffering capacity as compared to BAA or BAP1A1. It is reported that secondary and tertiary amines provide the endosomolytic activity due to its buffering effect; particularly, tertiary amines might also contribute to membrane disruption in the more acidic environment of lysosomes.40 As BAA and BAP1A1 possess full or partial AEPZ ending caps which contain tertiary amines, the relatively higher buffering capacity is probably associated with the high density of tertiary amines in the whole architecture. It is noted that all of the PAA polymers showed lower buffering capacity as compared to 25 kDa PEI, a wellknown transfection agent for its strong proton-sponge effect in the pH range of 7.4 to 5.1. For example, while it was calculated that PEI was capable of binding 22 μmol of proton from pH 7.4 to 5, BAA, however, could only bind ca. 14 μmol of proton within this pH range. The low buffering capacity of PAA polymers is because of protonation at the last step of synthesis. Although the partial protonation is favorable in increasing the water solubility of PAAs, it prevents the protonated amines from further protonation when the polyplexes are in the acidic endosomes. This also explains with reason why all PAA polymers require a much higher amount to achieve an optimal

Figure 5. pDNA release from BAA polyplexes from 0 to 5 h in the presence of 0, 10 μM, and 10 mM DTT. The release DNA was quantified by PicoGreen fluorescence assay. Results are expressed as mean and standard deviation (n = 3).

polyplex nanostructure during the entire experimental period. Similarly, when BAA polyplexes were exposed to10 μM DTT, which is close to extracellular GSH concentration, there was also no indication of DNA release. At the DTT concentration of 10 mM, initial burst release was detected in the first 2 h, and the released DNA percentage increased significantly to 90% within this period. After two hours, the release of pDNA from G

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 7. Relative cell viability assay in (A) HEK293, (B) COS-7, (C) MCF-7, and (D) Hep G2 cell lines as a function of polymers concentration. The cells were incubated with PAA polymers at various concentrations for 24 h in a serum-containing medium. Cell viability was determined by MTT assay and expressed as a percentage of control, the untreated cells. Data represent mean ± SD (n = 4).

transfection ratio, which will be further discussed in the section about transfection. In Vitro Cytotoxicity of PAA Polymers and Polyplexes. The cytotoxicity of gene vectors has great influence on the transfection efficiency for DNA delivery. Many factors including molecular weight, charge density, type of amines, polymer structure, and biodegradability may affect cytotoxicity of the vectors. It was hypothesized that cytotoxicity was related to gene transfection efficiency, and this might be caused by electrostatic interactions with negatively charged glycocalyx on the cell surface. The presence of primary, secondary, and tertiary amines, which are usually charged under physiological conditions are believed to induce the cytotoxicity of gene vectors.41 Figure 7 shows the cell viability as a function of polymer concentration using MTT assay in COS-7, HEH293, MCF-7 and Hep G2 cell lines. PEI showed a very strong dosedependent effect on cytotoxicity in all cell lines, and the cell viability dropped to 20% above the PEI concentration of 125 μg/mL. The cytotoxicity of PAA polymers, however, were much less dependent on the increasing polymer concentration in the entire concentration range from 16 to 500 μg/mL, indicating a very low or even neglectable cytotoxicity of these polymers. BAA showed slight toxicity in HepG2, MCF-7, and COS-7 cell lines when the concentration became higher than 250 μg/mL, inducing a total of 10−20% cell death. It should be pointed out that there is almost no distinguished difference between BAA and other polymers in terms of dose-dependent cytotoxicity. Similar trends were observed for other PAA polymers in these cells. The low cytotoxicity of PAA polymers may be attributed to low amino density in the PAA core and biocompatible terminals in the peripherals that have imparted biocompatible characteristics to the cationic carriers. The cytotoxicity of the PAA polyplexes was also evaluated as a

function of polymer/DNA weight ratio. As presented in Figure S3, it was found that the cytotoxicity of PAA polyplexes was merely affected by increasing the weight ratio of polyplexes. Whereas BAAP, BAP350, and BAP750 showed almost no cytotoxicity in the entire range of tested weight ratio, BAA and BAP1A1 polyplexes exhibited slightly lower relative cell viability in comparison with other PAA polyplexes at high weight ratio. This is probably associated with the relatively higher amino density of BAA and BAP1A1 in comparison with other PAA polymers. In Vitro Gene Transfection of PAA Polyplexes. In order to evaluate the effect of different terminals on the transfection activity, transfection efficiency of PAA polyplexes was first assessed using luciferase as a marker gene in the presence of serum. The luciferase expression of PAA-mediated gene transfection was quantified by the means of a luciferase activity assay compared with commercially available PEI (25 kDa). Gene transfection mediated by PAA polymers was carried out in the four different kinds of cell lines as described earlier. It was found that luciferase expression of PAA-mediated gene transfection was greatly dependent on the cell type and weight ratio of the polyplexes (Figure S4). The conversion of polymer/DNA weight ratio to nitrogen/phosphorus (N/P) ratio and the optimal weight ratio for gene transfection were presented in Table S2 and Table S3, respectively. Generally, the gene transfection efficiency increased with the increasing weight ratios. At the optimal weight ratio, transfection mediated by BAA showed the highest efficiency in all cell lines, whereas BAP750 showed the poorest transfection activity (Figure 8). The high transfection of BAA should attribute to AEPZ terminals bearing primary amines which are responsible for DNA condensation and tertiary amines that facilitate endosomal escape. The low transfection activity of BAP750 is H

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

GSH level. GSH has been found to be elevated in a number of carcinoma cell lines including Hep G2 and MCF-7,45,46 and BSO is well-documented to reduce GSH level by inhibiting GSH biosynthesis in both cell lines. The present study investigates whether inhibition of GSH will significantly affect the transfection properties. The effect of BSO concentration on the GSH inhibition level was first examined. As shown in Figure S6, the concentration of BSO at 0.2 mM was able to drastically deplete the GSH in both MCF-7 and Hep G2 cells by 3-fold. Any further increase in BSO concentration did not result in any more drastic depletion of GSH in both cell lines. In addition, BSO concentration up to 1 mM did not cause significant toxicity over these cell lines (Figure S7). In light of significant toxicity and other side effects associated with BSO, the lowest BSO concentration (0.2 mM) that was effective in inhibiting GSH synthesis was used for the subsequent investigations. As shown in Figure 9A, in the case of both carcinoma cell lines, transfection mediated by nonredox-sensitive PEI in these

Figure 8. In vitro luciferase gene expression in various types of cell lines transfected with PAA polyplexes at optimal polymer/DNA weight ratio. The weight ratios of BAA, BAAP, BAPP1A1, BAP350, BAP750/DNA are 90, 90, 45, 90, 90 in HEK293 cells, 90, 60, 45, 45, 90 in COS-7 cells, 45, 60, 45, 30, 90 in MCF-7 cells, 60, 60, 45, 45, 45 in Hep G2 cells, respectively. PEI/DNA complexes at weight ratio of 2 (N/P ratio of 15) was used as a control. Data represent mean ± SD (n = 3, Student’s t test, *P < 0.05).

probably due to the PEG shielding effect, which greatly hampered the charge-mediated cellular uptake. When the PAA was end-capped with APD, the transfection efficiency was also dramatically decreased in HEK293, COS-7 and MCF-7 cells. Thus, it is evident that end-capping with alcohol-bearing molecules for hyperbranched PAA is generally not favorable for transfection in vitro, although the similar structure of 4aminobutanol or 2-aminoethanol was used to seal hyperbranched PAA in the early study.22 Surprisingly, BAAP showed comparable transfection activity as BAA in Hep G2 cells at their optimal polymer/DNA ratio. It was suggested by Reineke and co-workers that hydroxyl stereochemistry and the number of amine units within poly(glycoamidoamine)s affect the gene delivery efficiency, and this effect was also found to be cell-type dependent.42 However, it is not clear whether the improved transfection activity of BAAP in Hep G2 cell lines is due to the hydroxyl stereochemistry within BAAP, and further investigations are essential to clarify this phenomenon. In the case of BAP1A1 partially terminated with PEG, it showed comparable or higher transfection activity as compared to that of BAP350 fully terminated with PEG. It should be pointed out that all PAA polymers requires a much higher PAA/DNA weight ratio for transfection as compared to PEI, which was attributed to two reasons in our point of view. First, PEI showed much higher charge density over PAA polymers. Second, PAAs possess the poor buffering capacity at pH 7.4−5.0 and require a large amount of polymers within polyplexes to aid polyplexes for effective endosomal escape. The above results demonstrate the terminal structure of PAA plays a critical role in achieving high efficiency of PAA-mediated transfection. Transfection mediated by BAA in reduced serum condition was also carried out as shown in Figure S5. In comparison with transfection in serum-free condition, it was found that the presence of serum (10% FBS) could further enhance the transfection activity of BAA, which was probably due to the high cell viability in the presence of serum.22 The Effect of BSO on Transfection Efficiency. BSO is a specific γ-glutamylcysteine synthetase inhibitor that blocks the rate-limiting step of glutathionine (GSH) biosynthesis. Intracellular GSH pool in both cultured cells and in whole animals can be depleted in the presence of BSO.43,44 Therefore, artificial modulation of GSH levels can be achieved by using BSO to inhibit GSH synthesis, thus reducing or depleting the cellular

Figure 9. (A) In vitro luciferase gene expression in Hep G2 and MCF7 cells transfected with BAA in the presence or absence of 0.2 mM BSO. Nonredox sensitive PEI polyplex at polymer/DNA ratio of 2 was used as a control. (B) Cell viability of Hep G2 and MCF-7 cells transfected with BAA/DNA complexes in the presence or absence of 0.2 mM BSO. All data represent mean ± SD (n = 3, Student’s t test, *P < 0.05, **P < 0.01, **P < 0.005).

cells treated with 0.2 mM of BSO gave a similar level of luciferase expression as the cells without BSO treatment. However, transfection efficiency dropped nearly by 1 order of magnitude in Hep G2 cells after BSO treatment. There are two possible reasons associated with decreased transfection efficiency. First, it has been widely accepted that DNA release triggered by intracellular GSH at the cytoplasm and nucleus would become difficult due to the insufficient reduction of disulfide bonds because of the low GSH concentration.47 Second, due to the slow degradation rate as a result of a low I

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

Figure 10. (A) The confocal microscopy images of Hep G2 cells transfected with BAA/pEGFP complexes in the presence or absence of BSO at different transfection time (red: RDM-pDNA; green: GFP; blue: DAPI). The white bar represents 20 μm. (B) Cellular uptake and (C) GFP expression of BAA/RDM-pEGFP complexes evaluated by flow cytometry analysis. Results are expressed as mean and standard deviation (n = 3).

GSH level, large amounts of nondegraded polymers will accumulate inside the cells and cause a much higher cytotoxicity, which in turn influences the transfection efficiency. To prove our hypothesis, we further compared cytotoxicity profiles mediated by BAA polyplexes in the presence or absence of 0.2 mM BSO (Figure 9B). At a dose level of 0.5 μg DNA/ 104 cells, the cytotoxicity of BAA polyplexes is only slightly higher after BSO treatment. When increasing the DNA dose level to 1.0 μg DNA/104 cells, it was found that the difference between cells with BSO treatment and cells without BSO is more obvious. While the cell viability of BAA polyplexes in Hep G2 cells dropped from 68% to 36% after BSO treatment, the cytotoxicity induced by BAA polyplexes in the presence of BSO resulted in a significant decrease of cell viability of MCF-7 cells from 87% to 55%. Therefore, it is clear that the inhibition of GSH synthesis would also prevent the degradation of BAA, leading to significant cytotoxicity as a result of accumulation of HMW BAA intracellularly. Reduced GFP expression mediated by BAA polyplexes in the presence of BSO at Hep G2 cells was also confirmed by confocal laser scanning microscopy (CLSM). As can been seen in Figure 10A, GFP expression in both BSO-treated and BSOfree cells was very weak (below 5%) at 2 h post-transfection, and almost no green fluorescence can be visualized under the confocal microscope. In BSO-free Hep G2 cell lines, the GFP expression became strong at 10 h post-transfection, reaching the highest level at 24 h. A similar trend of increasing GFP expression was also observed in BSO-treated over time. Despite a comparable level of cellular uptake at 10 or 24 h posttransfection (Figure 10B), the percentage of GFP cells with BSO treatment was lower at the same time point compared to

that of cells without BSO treatment (Figure 10C). For example, at 24 h post-transfection, the percentage of GFP positive cells was found to be 25% without BSO treatment, whereas it decreased to 18% with BSO treatment. This also strongly indicates that transfection activity of PAAs is strongly dependent on intracellular GSH level.

■. CONCLUSIONS Hyperbranched PAAs with tertiary amino cores and different terminal groups were synthesized. Although all PAA polymers possess the same thiol-responsive core, the difference in the terminal structures results in a definite distinction in terms of biophysical properties and transfection efficiency in vitro. While all PAAs were able to condense pDNA into nanoparticles of a diameter of 50−200 nm with a positive surface charge at polymer/DNA weight ratio higher than 20, PAA terminated by AEPZ (BAA) exhibit the highest transfection activity in all the tested cell lines. Rapid degradation of BAA polyplexes, which is initiated by the cleavage of the disulfide bonds, can be detected within 2 h in the presence of 10 mM DTT. Furthermore, intracellular GSH level modulated by BSO can significantly affect the transfection efficiency and cytotoxicity of BAA polyplexes. This study has demonstrated the degradation of disulfide bonds in the PAA core plays an important role in achieving high transfection efficiency and low cytotoxicity. The terminal structure in PAA shell significantly affects gene transfection activity in vitro and PAAs with tertiary amino cores are promising for safe and efficient gene delivery. J

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

■

Article

(14) Wu, D.; Liu, Y.; Jiang, X.; Chen, L.; He, C.; Goh, S. H.; Leong, K. W. Biomacromolecules 2005, 6, 3166−3173. (15) Arote, R. B.; Jiang, H. L.; Kim, Y. K.; Cho, M. H.; Choi, Y. J.; Cho, C. S. Expert Opin. Drug Delivery 2011, 8, 1237−1246. (16) Christensen, L. V.; Chang, C. W.; Kim, W. J.; Kim, S. W.; Zhong, Z.; Lin, C.; Engbersen, J. F.; Feijen, J. Bioconjugate Chem. 2006, 17, 1233−1240. (17) Wang, X.; He, Y.; Wu, J.; Gao, C.; Xu, Y. Biomacromolecules 2010, 11, 245−251. (18) Wang, R.; Zhou, L.; Zhou, Y.; Li, G.; Zhu, X.; Gu, H.; Jiang, X.; Li, H.; Wu, J.; He, L.; Guo, X.; Zhu, B.; Yan, D. Biomacromolecules 2010, 11, 489−495. (19) Yu, Z. Q.; Yan, J. J.; You, Y. Z.; Zhou, Q. H. Int. J. Nanomed. 2012, 7, 5819−5832. (20) Lin, C.; Blaauboer, C. J.; Timoneda, M. M.; Lok, M. C.; van, S. M.; Hennink, W. E.; Zhong, Z.; Feijen, J.; Engbersen, J. F. J. Controlled Release 2008, 126, 166−174. (21) Manickam, D. S.; Li, J.; Putt, D. A.; Zhou, Q. H.; Wu, C.; Lash, L. H.; Oupicky, D. J. Controlled Release 2010, 141, 77−84. (22) Martello, F.; Piest, M.; Engbersen, J. F.; Ferruti, P. J. Controlled Release 2012, 164, 372−379. (23) Lin, C.; Zhong, Z.; Lok, M. C.; Jiang, X.; Hennink, W. E.; Feijen, J.; Engbersen, J. F. J. Controlled Release 2006, 116, 130−137. (24) Chen, J.; Wu, C.; Oupicky, D. Biomacromolecules 2009, 10, 2921−2927. (25) Lin, C.; Engbersen, J. F. J. Controlled Release 2008, 132, 267− 272. (26) Meng, F.; Hennink, W. E.; Zhong, Z. Biomaterials 2009, 30, 2180−2198. (27) Wu, D.; Liu, Y.; Jiang, X.; He, C.; Goh, S. H.; Leong, K. W. Biomacromolecules 2006, 7, 1879−1883. (28) Liu, Y.; Wu, D.; Ma, Y.; Tang, G.; Wang, S.; He, C.; Chung, T.; Goh, S. Chem. Commun. (Cambridge, U.K.) 2003, 2630−2631. (29) Wu, D. C.; Loh, X. J.; Wu, Y. L.; Lay, C. L.; Liu, Y. J. Am. Chem. Soc. 2010, 132, 15140−15143. (30) van de, W. P.; Cherng, J. Y.; Talsma, H.; Crommelin, D. J.; Hennink, W. E. J. Controlled Release 1998, 53, 145−153. (31) Ping, Y.; Liu, C. D.; Tang, G. P.; Li, J. S.; Li, J.; Yang, W. T.; Xu, F. J. Adv. Funct. Mater. 2010, 20, 3106−3116. (32) Ferruti, P.; Manzoni, S.; Richardson, S. C. W.; Duncan, R.; Pattrick, N. G.; Mendichi, R.; Casolaro, M. Macromolecules 2000, 33, 7793−7800. (33) Mongondry, P.; Bonnans-Plaisance, C.; Jean, M.; Tassin, J. F. Macromol. Rapid Commun. 2003, 24, 681−685. (34) Ping, Y.; Liu, C.; Zhang, Z.; Liu, K. L.; Chen, J.; Li, J. Biomaterials 2011, 32, 8328−8341. (35) Nimesh, S.; Goyal, A.; Pawar, V.; Jayaraman, S.; Kumar, P.; Chandra, R.; Singh, Y.; Gupta, K. C. J. Controlled Release 2006, 110, 457−468. (36) Sung, S. J.; Min, S. H.; Cho, K. Y.; Lee, S.; Min, Y. J.; Yeom, Y. I.; Park, J. K. Biol. Pharm. Bull. 2003, 26, 492−500. (37) Wang, D. A.; Narang, A. S.; Kotb, M.; Gaber, A. O.; Miller, D. D.; Kim, S. W.; Mahato, R. I. Biomacromolecules 2002, 3, 1197−1207. (38) Ogris, M.; Steinlein, P.; Carotta, S.; Brunner, S.; Wagner, E. AAPS PharmSci. 2001, 3, E21. (39) Ogris, M.; Steinlein, P.; Kursa, M.; Mechtler, K.; Kircheis, R.; Wagner, E. Gene Ther. 1998, 5, 1425−1433. (40) Cho, Y. W.; Kim, J. D.; Park, K. J. Pharm. Pharmacol. 2003, 55, 721−734. (41) Fischer, D.; Li, Y.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. Biomaterials 2003, 24, 1121−1131. (42) Liu, Y.; Reineke, T. M. J. Am. Chem. Soc. 2005, 127, 3004−3015. (43) Drew, R.; Miners, J. O. Biochem. Pharmacol. 1984, 33, 2989− 2994. (44) Armstrong, J. S.; Steinauer, K. K.; Hornung, B.; Irish, J. M.; Lecane, P.; Birrell, G. W.; Peehl, D. M.; Knox, S. J. Cell Death Differ. 2002, 9, 252−263. (45) Huang, Z. Z.; Chen, C.; Zeng, Z.; Yang, H.; Oh, J.; Chen, L.; Lu, S. C. FASEB J. 2001, 15, 19−21.

ASSOCIATED CONTENT

S Supporting Information *

Full 13C NMR spectra, molecular weight of PAA polymers, the effect of weight ratio of polymer/DNA on transfection efficiency, the optimal transfection polymer/DNA weight ratio in different cell lines, and the conversion of polymer/ DNA weight ratio to nitrogen/phosphorus (N/P) ratio are available in the Supporting Information. In addition, cell viability as a function of different weight ratio of polymer/DNA complexes, the effect of serum on BAA-mediated transfection, the effect of BSO on the intracellular GSH level, and relative cell viability as a function of BSO concentration are also included in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +65 6874 8105; Fax: +65 6872 7528; E-mail: ye-liu@ imre.a-star.edu.sg. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The financial support is from A*STAR under the JCO program and the Singapore−China Joint Research Programme. The authors greatly acknowledged Mr. Zhang Weian and Ms. Lee Shu Ying from the Yong Loo Lin School of Medicine, National University of Singapore, for their skillful assistance on the confocal laser scanning microscope study, and Ms. Peng Shishi Cherrie from Nanyang Technological University for her assistance in revising the manuscript.

■

REFERENCES

(1) Giacca, M.; Zacchigna, S. J. Controlled Release 2012, 161, 377− 388. (2) Elsabahy, M.; Nazarali, A.; Foldvari, M. Curr. Drug Delivery 2011, 8, 235−244. (3) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259−302. (4) Viola, J. R.; El-Andaloussi, S.; Oprea, I. I.; Smith, C. I. Expert Opin. Drug Delivery 2010, 7, 721−735. (5) Koping-Hoggard, M.; Tubulekas, I.; Guan, H.; Edwards, K.; Nilsson, M.; Varum, K. M.; Artursson, P. Gene Ther. 2001, 8, 1108− 1121. (6) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297−7301. (7) Kwoh, D. Y.; Coffin, C. C.; Lollo, C. P.; Jovenal, J.; Banaszczyk, M. G.; Mullen, P.; Phillips, A.; Amini, A.; Fabrycki, J.; Bartholomew, R. M.; Brostoff, S. W.; Carlo, D. J. Biochim. Biophys. Acta 1999, 1444, 171−190. (8) Bellocq, N. C.; Pun, S. H.; Jensen, G. S.; Davis, M. E. Bioconjug. Chem. 2003, 14, 1122−1132. (9) Malik, N.; Wiwattanapatapee, R.; Klopsch, R.; Lorenz, K.; Frey, H.; Weener, J. W.; Meijer, E. W.; Paulus, W.; Duncan, R. J. Controlled Release 2000, 65, 133−148. (10) Liu, W. M.; Liu, M.; Xue, Y. N.; Peng, N.; Xia, X. M.; Zhuo, R. X.; Huang, S. W. J. Biomed. Mater. Res. A 2012, 100, 872−881. (11) Lunsford, L.; McKeever, U.; Eckstein, V.; Hedley, M. L. J. Drug Target 2000, 8, 39−50. (12) Liu, Y.; Wu, D. C.; Zhang, W. D.; Jiang, X.; He, C. B.; Chung, T. S.; Goh, S. H.; Leong, K. W. Angew. Chem., Int. Ed. Engl. 2005, 44, 4782−4785. (13) Wong, K.; Sun, G.; Zhang, X.; Dai, H.; Liu, Y.; He, C.; Leong, K. W. Bioconjugate Chem. 2006, 17, 152−158. K

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules

Article

(46) Lewis-Wambi, J. S.; Kim, H. R.; Wambi, C.; Patel, R.; Pyle, J. R.; Klein-Szanto, A. J.; Jordan, V. C. Breast Cancer Res. 2008, 10, R104. (47) Kim, T. I.; Kim, S. W. React. Funct. Polym. 2011, 71, 344−349.

L

dx.doi.org/10.1021/bm400460r | Biomacromolecules XXXX, XXX, XXX−XXX