Bio-Medical Materials and Engineering 24 (2014) 673–682 DOI 10.3233/BME-130855 IOS Press
673
Dextranation of Bioreducible Cationic Polyamide for Systemic Gene Delivery Chao Lina,* †, Yanyan Songa, b†, Bo Loua, b and Peng Zhaoa,* a
The Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Tongji University, Shanghai, 200092, P.R.. China. b School of Life Sciences and Technology, Tongji University, Shanghai, 200092, P.R.. China
Abstract. A dextranated, bioreducible cationic polyamide was designed and employed for non-viral gene delivery in vitro and in vivo. Initially, a new bioreducible cationic polyamide with p-nitrophenyl ester terminal group (denoted as SSBAP) was synthesized by polycondensation reaction of an excess amount of bis-(p-nitrophenyl)-3, 3’-dithiodipropanoate and 1, 4-bis(3aminopropyl)piperazine. The SSBAP was then chemically conjugated with 5kDa amino-terminated dextran to yield dextranSSBPA-dextran triblock copolymer (denoted as Dex-SSBAP-Dex). This copolymer was capable of binding genes to form nanoscale polyplexes with a near-neutral surface charge. Moreover, a sufficient gene release from the polyplexes in response to an intracellular reducing environment was observed. In vitro transfection against MCF-7 and SKOV-3 cells showed that Dex-SSBAP-Dex copolymer effectively transfected the cells with comparable efficiency to that of 25kDa branched or linear polyethylenimine as positive controls. Besides, intravenous administration of the copolymer-based polyplexes in nude mice afforded detectable gene expression largely in the lung. Importantly, the copolymer revealed low cytotoxicity in vitro, as determined by AlamarBlue assay, and caused no death of the mice. Dextranated, bioreducible cationic polyamide holds high potential as a non-viral vector for gene delivery. Keywords: Dextran, disulfide, gene delivery, transfection, in vivo
1. Introduction The development of safe and highly potent gene delivery vectors is a big challenge for successful gene therapy. Although recombinant viral vectors are popular for clinical gene therapy, their safety issues such as immunogenicity, mutagenesis and oncogenicity remain controversial [1, 2]. Alternatively, non-viral vectors such as cationic polymers have received much attention in the past few years. As compared to viral vectors, cationic polymers have a few advantages such as low immune response, handy synthesis and modification, unlimited gene-carrying capacity, and large-scale production [3]. A lot of cationic polymers including polyethylenimine (pEI) and poly(L-lysine) have thus been studied widely for in vitro gene delivery [4]. These polymers can spontaneously self-assemble with genes to form nanoscale and positively-charged polyplexes that are capable of delivering the genes into the cellular nucleus, causing detectable transfection efficiency [5]. However, these traditional polymer systems are not yet satisfied for clinical trials in part due to their low transfection efficiency and/or high *
Corresponding author. E-mail: [email protected] (CL), [email protected] (PZ) † The authors contribute equally as co-first author. 0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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C. Lin et al. / Dextranation of bioreducible cationic polyamide for systemic gene delivery
cytotoxicity [6]. Furthermore, inherent extracellular gene delivery barriers impose restriction on further implement of cationic polyplexes for in vivo gene delivery [7]. For example, after intravenous (i.v.) injection, cationic polyplexes are prone to aggregation due to their non-specific interactions with serum and blood cells, causing dangerous blood clotting. Moreover, cationic polyplexes are rapidly eliminated by the reticuloendothelial system due to opsonization of the polyplexes during blood circulation [8]. Thus, systemic transfection efficacy of cationic polyplexes by i.v. administration is rather inefficient. Different methods have been developed to design charge-shielded polyplexes for systemic gene delivery, in order to circumvent the extracellular gene delivery barriers. One of the most widely used approaches for the polyplexes involves chemical modification of cationic polymers with poly (ethylene glycol) (PEG). PEGylation method affords PEGylated cationic polymers to condense genes into PEGylated polyplexes. It is known that, as opposed to cationic polyplexes, PEGylated polyplexes display a prolonged gene delivery profile due to their near-neutral charge surface and improved colloidal stability in physiological conditions [9]. Alternatively, chemical coupling of dextran to cationic polymers is an effective method to gain charge-shielded polyplexes. Dextran is a nonionic, hydrophilic polysaccharide. This neutral polymer offers dextranated nanoparticles with “stealth” property for longterm circulation since their neutral surface charge may weaken opsonization process [10, 11]. In the past two decades, a few dextranated polycations based on oligoamines [12, 13] and low-molecularweight PEIs [14, 15] have been studied for non-viral gene delivery in vitro. However, in vivo transfection activity of these cationic dextran systems has rarely been reported. For example, Domb et al. showed that dextran-spermine polycation induced high gene expression by intramuscular or intranasal administration, but no transfection efficacy by intravenous (i.v.) administration was found[16]. In the past five years, the researches on the design of biodegradable cationic polymers for non-viral gene delivery have been increasing. Particularly, disulfide-based (bioreducible) cationic polymers have been paid more and more attention [17]. For example, a few types of bioreducible PEI analogues were prepared by chemical coupling of oligoamines with disulfide-containing reagents [18, 19]. A number of bioreducible poly(amido amine)s were synthesized via Michael-addition reaction of cystaminebisacryamide and different amines [20-22]. It is well known that disulfide bond is chemically stable in physiological conditions , but is degradable by the glutathione present in the cell interior [23]. For that reason, bioreducible cationic polymers are intracellularly biodegradable due to the cleavage of disulfide bond, in turn causing a sufficient unpacking of polyplexes and efficient gene release. This unique property offers bioreducible cationic polymers with higher transfection efficiency as compared to their counterparts without disulfide bond. This intracellular biodegradation also endows bioreducible cationic polymers with low cytotoxicity in vitro. Thus, the current context indicates that bioreducible cationic polymers have a promise for low-toxic and efficient polymeric gene delivery vectors [24]. The purpose of this study is to generate a dextranated, bioreducible polycation for systemic gene delivery by i.v. administration. A new bioreducible cationic polyamine having p-nitrophenyl ester ending group (denoted as SSBAP) was synthesized by polycondensation reaction of bis-(p-nitrophenyl)-3, 3’dithiodipropanoate and 1, 4-bis(3-aminopropyl)piperazine (Figure 1a). Subsequently, dextranation of the SSBAP was conducted with 5kDa amino-terminated dextran to generate dextran-SSBAP-dextran copolymer. We hypothesized that this copolymer is able to condense genes into nanoscale polyplexes with near neutral surface charge and mediate efficient gene delivery in vitro and in vivo. Chemical and biophysical properties of this copolymer were studied in terms of gene binding behavior, colloidal stability in physiological conditions, and gene release profile in an intracellular reducing environment. In vitro transfection activity and cytotoxicity of the copolymer were evaluated in MCF-7 and SKOV-3
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cells. Moreover, in vivo transfection efficacy induced by the polyplexes of the copolymer was evaluated by intravenous administration in nude mice. 2. Materials and Methods 2.1. Materials Dextran (Mw=5kDa), pyridine, dithiothreitol (DTT), triethylamine (TEA), p-nitrophenol, anhydrous dimethyl sulfoxide (DMSO), p-nitrophenol, branched polyethylenimine (B-pEI, Mw=25kDa) were ordered from Sigma-Aldrich, Linear polyethylenimine (L-pEI, Mw=22kDa) from Polysciences, INC (USA). 3, 3’-dithiodipropionic acid (DTPA), 1, 4-bis(3-aminopropyl)piperazine (BAP) and thionyl chloride from AlfaAssar. Fetal bovine serum (FBS), McCoy's 5A medium, Dulbecco's Modified Eagle Medium (DMEM) and phosphate buffered saline (PBS) from GIBCO, pCMV-eGFP and pCMV-Luc plasmid from plasmid factory (Germany), and luciferase assay kit and cell lysis buffer from Promega. 2.2. Monomer synthesis and polymer preparation Bis-(p-nitrophenyl)-3,3’-dithiodipropionate (DTPA) was synthesized according to the method introduced in reference [25]. Firstly, a mixture of 3, 3’-dithiodipropionic acid (5.0 g, 0.024mol) and thionyl chloride (20.0 mL) in a round-bottom flask was heated under reflux at 85 °C for 4 h. Unreacted thionyl chloride was removed by rotary evaporation to give dithiodipropionic chloride. Secondly, dithiodipropionic chloride (0.024 mol) in acetone (50 mL) was added drop-wise to a mixture of p-nitrophenol (0.051 mol) and TEA (0.072 mol) in cooled acetone (100 mL) over 2 h at 0 °C and the mixture was stirred at room temperature overnight. The mixture was poured into distilled water (800 mL) and the precipitate was collected and finally obtained by re-crystallization in ethyl acetate (Yield: 2.4 g). 1H NMR (CCl3): 3.1 (s, 8H, -CH2CH2SS CH2CH2-), 8.3 and 7.3 (aromatic protons). Synthesis of amino-terminated dextran (Dex-NH2): Dex-NH2 was synthesized according to the method provided in previous references [26]. In brief, dextran was reacted with N-Boc-1, 4diaminobutane in the presence of sodium cyanoborohydride to modify the terminal group of dextran. Next, t-butyloxycarbonyl group was removed with TFA. In a typical experiment procedure, dextran (1 g) in ultrapure water (6 mL) was reacted with N-Boc-1, 4-diaminobutane (0.4 g) for 2 h under nitrogen protection. NaBH3CN (0.4 g) was then added in the reaction by six portions. The reaction mixture was stirred at room temperature for 3 days. The pH value of the solution was adjusted to pH 7 with 2 M HCl. Boc-aminated dextran (denoted as Dex-C4-NHBoc) was obtained by ultrafiltration purification (MWCO 1000) and isolated as white powder after freeze-drying (Yield: 0.6 g). 1H NMR (D2O): 1.31.4 (9H, -C(CH3)3), 1.4-1.7 (4H, -NH-CH2-C2H4-CH2-NHBoc), 3.0 (4H, -NH-CH2-C2H4-CH2-NHBoc), 3.2-4.1 (dextran glucosidic protons), 5.0 (dextran anomeric proton). Dex-C4-Boc (0.6 g) was dissolved in water (10 mL) and TFA (1.5 mL), and stirred overnight under nitrogen protection. The DextranNH2 was obtained by ultrafiltration purification (MWCO 1000) and freeze-dried (Yield: 0.4 g). 1H NMR (D2O): 1.5-1.6 (-NH-CH2-C2H4-CH2-NH2), 3.0 (-NH-CH2-C2H4-CH2-NH2), 3.2-4.1 (dextran glucosidic protons), 4.9 (dextran anomeric proton). Synthesis of dextran-coupled, bioreducible copolymer (Dex-SSBAP-Dex): The copolymer was prepared by a two-step procedure as shown in Figure 1a. Cationic polyamide with p-nitrophenyl ester terminal group (SSBAP) was prepared at the DTPA/BAP ratio of 1/0.9. In detail, the SSBAP with a theoretical average-number polymerization degree of 19 was synthesized by stirring a mixture of
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DTPA (1.10 mmol) and BAP (0.99 mmol) in anhydrous DMSO (7 ml) in a reaction flask at 40 ºC under nitrogen atmosphere, to yield SSBAP. After 72 h reaction, an excess of Dex-NH2 (0.25 g) was added and the mixture was stirred for another 48 h. The resulting polymer was made into white powder after ultrafiltration purification (MWCO 10000) and freezing-dry (Yield: 0.28 g). 1H NMR (D2O): 1.8 (-N-CH2-CH2-CH2-NNCO-), 2.5 (-N-CH2-CH2-CH2-NNCO-), 2.8 (-N-CH2-CH2-CH2-NNCO-), 3.1 (-CH2CH2SS CH2CH2-), 3.2-3.9 (dextran glucosidic protons, piperazine ring protons), 4.9 (dextran anomeric proton). 2.3 Chemical and biophysical Characterization 1
H NMR (400 MHz) spectra were recorded on a Varian Inova spectrometer (Varian, USA). The signals of solvent residues were used as reference and were set at 4.79 for D2O and 7.26 for CCl3 The polyplexes of Dex-SSBAP-Dex at different nitrogen/phosphate (N/P) ratios were prepared by adding the polymer solution (800 L, different concentrations in 20 mM HEPES buffer at pH 7.4) to DNA (200 L of 75 g/mL in 20 mM HEPES buffer at pH 7.4), followed by vortexing for 5 s and incubating at room temperature for 30 min. Particle sizes and surface charges of the polyplexes were measured at 25 ºC with Nanosizer NS90 (Malvern, UK). To evaluate colloidal stability, saline solution was added to give a final salt concentration of 150 mM. The resulting polyplex solution was incubated at different time (0.5 and 4 h). The size of the polyplexes was measured by Nanosizer NS90. 2.4 Gel electrophoresis The polyplexes at different N/P ratios were made by adding 10 L of the polymer solution (varying concentrations in 20 mM HEPES at pH 7.4) to 10 L of DNA solution (80 g/mL in 20 mM HEPES), followed by vortexing for 5 s and the dispersions were incubated for 30 min at room temperature. Next, 10 L of HEPES buffer (as control) or the buffer containing DTT was added to set final DDT concentration of 10 mM, and the mixtures were incubated for another 30 min. After adding 3 L of 6× loading buffer (Fermentas), 10 L of this mixture was applied onto a 0.7% agarose gel with ethidium bromide (0.5 g/mL). After development of the gel, DNA was visualized using Tanon Gel Image system. 2.5 Cell culture and gene transfection in vitro MCF-7 and SKOV-3 cells (ATCC) were cultured in DMEM and McCoy's 5 medium, respectively, containing 10% FBS and 100 U/mL penicillin and streptomycin. Transfection experiments were performed using pCMV-GFP or pCMV-Luc reporter plasmid. The cells were plated in a 24-well plate (57×104 cells/well) and cultured in 0.5 mL of completed medium for at least 24 h until 60-70% cell confluence was reached. They were then washed twice with fresh 1×PBS buffer and incubated in the medium with or without 10% FBS for gene transfection. Next, the polyplexes at varying N/P ratios were prepared. In a typical transfection experiment of 24-well plate, the cells were treated with the polyplexes (1 g DNA) for 1 h at 37 °C in a 5% CO2-containing atmosphere. Then, the medium was replaced with completed medium and the cells were further incubated for another 47 h. A transfection formulation with B-pEI or L-pEI, prepared at an optimal N/P ratio of 8/1, was used as positive controls. All the transfection experiments were done in triplicates. GFP expression in transfected cells was determined by measuring GFP fluorescence intensity. In brief, the cells were washed twice with 1×PBS buffer and incubated with cell lysis buffer (200 L) at 4 ºC for 20 min. The cell lysate was collected and centrifuged to pellet cellular debris, and then 100 L of cell lysate was transferred in a 96-
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well plate to test fluorescence intensity of GFP with excitation and emission wavelengths of 488 and 520 nm, respectively (Thermo Scientific, USA). Background fluorescence or auto-fluorescence was also measured with untreated cells as a blank control. Total protein concentration in collected cell lysate from each well was estimated with BAC Protein Assay kit (Invitrogen) and normalized using a standard BSA curve. GFP expression was presented as GFP fluorescence intensity (FI) normalized to protein mass and shown as arbitrary units (a. u.)/mg protein. To determine luciferase gene expression, luciferase activity was analyzed using a standard luciferase assay kit (Promega) with a luminometer (Thermo Scientific, USA). Luciferase expression was shown as relative light unit (RLU)/mg protein. All data were given as mean values (standard deviations) of three experiments. 2.6 Cell viability assay MCF-7 and SKOV-3 cells (5 × 103 cells/well) were respectively seeded in a 96-well plate. When the confluence reached 70~80%, the medium was replaced with serum-free medium. The cells were then exposed in Dex-SSBAP-Dex at polymer concentrations in the range from 2 to 100 μg/mL. After 1 h incubation, the cells were cultured in completed medium for another 47 h. Cell viability was determined by Alamar Blue assay. In brief, the cells were washed with fresh PBS and 200 L of 1× Alamar Blue-RMPI 1640 medium was added to each well. After 4 h of incubation, 100 L of the medium in each well was transferred to a 96-well plate for reading optical density (OD) at emission wavelengths of 570 nm and 630 nm, respectively, using Multiscan MK3 plate reader (Thermo Scientific, USA). Cell viability was calculated by the equation: Cell viability (%)= (ODsample –OD0)/ODcontrol – OD0)×100, where ODsample, ODcontrol, and OD0 are the OD values of the medium of treated cells, the medium of untreated cells, and 1×AlamarBlue-RMPI 1640 medium, respectively. The value for untreated cells (as control) was taken as 100% cell viability. 2.7 In vivo gene transfection in nude mice Animal experiments were officially approved by the Institutional Animal Care and Use Committee of Tongji University. The 4-6 week male Balb/c nude mice were intravenously administrated via tail vein with 400 l of polyplexes of Dex-SSBAP-Dex containing 50 g of pCMV-Luc and 5% glucose at an N/P ratio of 10/1. After 24 h, the mice were sacrificed and their liver, lung, spleens and kidneys were dissected, washed with PBS and homogenized in passive lysis buffer prepared by mixing 1 mL of 5× passive lysis buffer (Promega), 200 l of 50 mM phenylmethylslfonyl fluoride (PMSF) in methanol and 100 l of protease inhibitor (Sigma). The homogenate was centrifuged at 12,000 × g for 10 min at 4 °C. Then, 10 l of the supernatant was mixed with 100 μl of luciferase assay reagent (Promega) to determine luciferase expression. Data were given as mean values (standard deviations) of five experiments and expressed as relative light unit (RLU)/g tissue. 2.8 Statistical analysis Comparisons between two samples were performed with the student’s t-test. Differences were considered to be statistically significant at P
673
Dextranation of Bioreducible Cationic Polyamide for Systemic Gene Delivery Chao Lina,* †, Yanyan Songa, b†, Bo Loua, b and Peng Zhaoa,* a
The Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Tongji University, Shanghai, 200092, P.R.. China. b School of Life Sciences and Technology, Tongji University, Shanghai, 200092, P.R.. China
Abstract. A dextranated, bioreducible cationic polyamide was designed and employed for non-viral gene delivery in vitro and in vivo. Initially, a new bioreducible cationic polyamide with p-nitrophenyl ester terminal group (denoted as SSBAP) was synthesized by polycondensation reaction of an excess amount of bis-(p-nitrophenyl)-3, 3’-dithiodipropanoate and 1, 4-bis(3aminopropyl)piperazine. The SSBAP was then chemically conjugated with 5kDa amino-terminated dextran to yield dextranSSBPA-dextran triblock copolymer (denoted as Dex-SSBAP-Dex). This copolymer was capable of binding genes to form nanoscale polyplexes with a near-neutral surface charge. Moreover, a sufficient gene release from the polyplexes in response to an intracellular reducing environment was observed. In vitro transfection against MCF-7 and SKOV-3 cells showed that Dex-SSBAP-Dex copolymer effectively transfected the cells with comparable efficiency to that of 25kDa branched or linear polyethylenimine as positive controls. Besides, intravenous administration of the copolymer-based polyplexes in nude mice afforded detectable gene expression largely in the lung. Importantly, the copolymer revealed low cytotoxicity in vitro, as determined by AlamarBlue assay, and caused no death of the mice. Dextranated, bioreducible cationic polyamide holds high potential as a non-viral vector for gene delivery. Keywords: Dextran, disulfide, gene delivery, transfection, in vivo
1. Introduction The development of safe and highly potent gene delivery vectors is a big challenge for successful gene therapy. Although recombinant viral vectors are popular for clinical gene therapy, their safety issues such as immunogenicity, mutagenesis and oncogenicity remain controversial [1, 2]. Alternatively, non-viral vectors such as cationic polymers have received much attention in the past few years. As compared to viral vectors, cationic polymers have a few advantages such as low immune response, handy synthesis and modification, unlimited gene-carrying capacity, and large-scale production [3]. A lot of cationic polymers including polyethylenimine (pEI) and poly(L-lysine) have thus been studied widely for in vitro gene delivery [4]. These polymers can spontaneously self-assemble with genes to form nanoscale and positively-charged polyplexes that are capable of delivering the genes into the cellular nucleus, causing detectable transfection efficiency [5]. However, these traditional polymer systems are not yet satisfied for clinical trials in part due to their low transfection efficiency and/or high *
Corresponding author. E-mail: [email protected] (CL), [email protected] (PZ) † The authors contribute equally as co-first author. 0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
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C. Lin et al. / Dextranation of bioreducible cationic polyamide for systemic gene delivery
cytotoxicity [6]. Furthermore, inherent extracellular gene delivery barriers impose restriction on further implement of cationic polyplexes for in vivo gene delivery [7]. For example, after intravenous (i.v.) injection, cationic polyplexes are prone to aggregation due to their non-specific interactions with serum and blood cells, causing dangerous blood clotting. Moreover, cationic polyplexes are rapidly eliminated by the reticuloendothelial system due to opsonization of the polyplexes during blood circulation [8]. Thus, systemic transfection efficacy of cationic polyplexes by i.v. administration is rather inefficient. Different methods have been developed to design charge-shielded polyplexes for systemic gene delivery, in order to circumvent the extracellular gene delivery barriers. One of the most widely used approaches for the polyplexes involves chemical modification of cationic polymers with poly (ethylene glycol) (PEG). PEGylation method affords PEGylated cationic polymers to condense genes into PEGylated polyplexes. It is known that, as opposed to cationic polyplexes, PEGylated polyplexes display a prolonged gene delivery profile due to their near-neutral charge surface and improved colloidal stability in physiological conditions [9]. Alternatively, chemical coupling of dextran to cationic polymers is an effective method to gain charge-shielded polyplexes. Dextran is a nonionic, hydrophilic polysaccharide. This neutral polymer offers dextranated nanoparticles with “stealth” property for longterm circulation since their neutral surface charge may weaken opsonization process [10, 11]. In the past two decades, a few dextranated polycations based on oligoamines [12, 13] and low-molecularweight PEIs [14, 15] have been studied for non-viral gene delivery in vitro. However, in vivo transfection activity of these cationic dextran systems has rarely been reported. For example, Domb et al. showed that dextran-spermine polycation induced high gene expression by intramuscular or intranasal administration, but no transfection efficacy by intravenous (i.v.) administration was found[16]. In the past five years, the researches on the design of biodegradable cationic polymers for non-viral gene delivery have been increasing. Particularly, disulfide-based (bioreducible) cationic polymers have been paid more and more attention [17]. For example, a few types of bioreducible PEI analogues were prepared by chemical coupling of oligoamines with disulfide-containing reagents [18, 19]. A number of bioreducible poly(amido amine)s were synthesized via Michael-addition reaction of cystaminebisacryamide and different amines [20-22]. It is well known that disulfide bond is chemically stable in physiological conditions , but is degradable by the glutathione present in the cell interior [23]. For that reason, bioreducible cationic polymers are intracellularly biodegradable due to the cleavage of disulfide bond, in turn causing a sufficient unpacking of polyplexes and efficient gene release. This unique property offers bioreducible cationic polymers with higher transfection efficiency as compared to their counterparts without disulfide bond. This intracellular biodegradation also endows bioreducible cationic polymers with low cytotoxicity in vitro. Thus, the current context indicates that bioreducible cationic polymers have a promise for low-toxic and efficient polymeric gene delivery vectors [24]. The purpose of this study is to generate a dextranated, bioreducible polycation for systemic gene delivery by i.v. administration. A new bioreducible cationic polyamine having p-nitrophenyl ester ending group (denoted as SSBAP) was synthesized by polycondensation reaction of bis-(p-nitrophenyl)-3, 3’dithiodipropanoate and 1, 4-bis(3-aminopropyl)piperazine (Figure 1a). Subsequently, dextranation of the SSBAP was conducted with 5kDa amino-terminated dextran to generate dextran-SSBAP-dextran copolymer. We hypothesized that this copolymer is able to condense genes into nanoscale polyplexes with near neutral surface charge and mediate efficient gene delivery in vitro and in vivo. Chemical and biophysical properties of this copolymer were studied in terms of gene binding behavior, colloidal stability in physiological conditions, and gene release profile in an intracellular reducing environment. In vitro transfection activity and cytotoxicity of the copolymer were evaluated in MCF-7 and SKOV-3
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cells. Moreover, in vivo transfection efficacy induced by the polyplexes of the copolymer was evaluated by intravenous administration in nude mice. 2. Materials and Methods 2.1. Materials Dextran (Mw=5kDa), pyridine, dithiothreitol (DTT), triethylamine (TEA), p-nitrophenol, anhydrous dimethyl sulfoxide (DMSO), p-nitrophenol, branched polyethylenimine (B-pEI, Mw=25kDa) were ordered from Sigma-Aldrich, Linear polyethylenimine (L-pEI, Mw=22kDa) from Polysciences, INC (USA). 3, 3’-dithiodipropionic acid (DTPA), 1, 4-bis(3-aminopropyl)piperazine (BAP) and thionyl chloride from AlfaAssar. Fetal bovine serum (FBS), McCoy's 5A medium, Dulbecco's Modified Eagle Medium (DMEM) and phosphate buffered saline (PBS) from GIBCO, pCMV-eGFP and pCMV-Luc plasmid from plasmid factory (Germany), and luciferase assay kit and cell lysis buffer from Promega. 2.2. Monomer synthesis and polymer preparation Bis-(p-nitrophenyl)-3,3’-dithiodipropionate (DTPA) was synthesized according to the method introduced in reference [25]. Firstly, a mixture of 3, 3’-dithiodipropionic acid (5.0 g, 0.024mol) and thionyl chloride (20.0 mL) in a round-bottom flask was heated under reflux at 85 °C for 4 h. Unreacted thionyl chloride was removed by rotary evaporation to give dithiodipropionic chloride. Secondly, dithiodipropionic chloride (0.024 mol) in acetone (50 mL) was added drop-wise to a mixture of p-nitrophenol (0.051 mol) and TEA (0.072 mol) in cooled acetone (100 mL) over 2 h at 0 °C and the mixture was stirred at room temperature overnight. The mixture was poured into distilled water (800 mL) and the precipitate was collected and finally obtained by re-crystallization in ethyl acetate (Yield: 2.4 g). 1H NMR (CCl3): 3.1 (s, 8H, -CH2CH2SS CH2CH2-), 8.3 and 7.3 (aromatic protons). Synthesis of amino-terminated dextran (Dex-NH2): Dex-NH2 was synthesized according to the method provided in previous references [26]. In brief, dextran was reacted with N-Boc-1, 4diaminobutane in the presence of sodium cyanoborohydride to modify the terminal group of dextran. Next, t-butyloxycarbonyl group was removed with TFA. In a typical experiment procedure, dextran (1 g) in ultrapure water (6 mL) was reacted with N-Boc-1, 4-diaminobutane (0.4 g) for 2 h under nitrogen protection. NaBH3CN (0.4 g) was then added in the reaction by six portions. The reaction mixture was stirred at room temperature for 3 days. The pH value of the solution was adjusted to pH 7 with 2 M HCl. Boc-aminated dextran (denoted as Dex-C4-NHBoc) was obtained by ultrafiltration purification (MWCO 1000) and isolated as white powder after freeze-drying (Yield: 0.6 g). 1H NMR (D2O): 1.31.4 (9H, -C(CH3)3), 1.4-1.7 (4H, -NH-CH2-C2H4-CH2-NHBoc), 3.0 (4H, -NH-CH2-C2H4-CH2-NHBoc), 3.2-4.1 (dextran glucosidic protons), 5.0 (dextran anomeric proton). Dex-C4-Boc (0.6 g) was dissolved in water (10 mL) and TFA (1.5 mL), and stirred overnight under nitrogen protection. The DextranNH2 was obtained by ultrafiltration purification (MWCO 1000) and freeze-dried (Yield: 0.4 g). 1H NMR (D2O): 1.5-1.6 (-NH-CH2-C2H4-CH2-NH2), 3.0 (-NH-CH2-C2H4-CH2-NH2), 3.2-4.1 (dextran glucosidic protons), 4.9 (dextran anomeric proton). Synthesis of dextran-coupled, bioreducible copolymer (Dex-SSBAP-Dex): The copolymer was prepared by a two-step procedure as shown in Figure 1a. Cationic polyamide with p-nitrophenyl ester terminal group (SSBAP) was prepared at the DTPA/BAP ratio of 1/0.9. In detail, the SSBAP with a theoretical average-number polymerization degree of 19 was synthesized by stirring a mixture of
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C. Lin et al. / Dextranation of bioreducible cationic polyamide for systemic gene delivery
DTPA (1.10 mmol) and BAP (0.99 mmol) in anhydrous DMSO (7 ml) in a reaction flask at 40 ºC under nitrogen atmosphere, to yield SSBAP. After 72 h reaction, an excess of Dex-NH2 (0.25 g) was added and the mixture was stirred for another 48 h. The resulting polymer was made into white powder after ultrafiltration purification (MWCO 10000) and freezing-dry (Yield: 0.28 g). 1H NMR (D2O): 1.8 (-N-CH2-CH2-CH2-NNCO-), 2.5 (-N-CH2-CH2-CH2-NNCO-), 2.8 (-N-CH2-CH2-CH2-NNCO-), 3.1 (-CH2CH2SS CH2CH2-), 3.2-3.9 (dextran glucosidic protons, piperazine ring protons), 4.9 (dextran anomeric proton). 2.3 Chemical and biophysical Characterization 1
H NMR (400 MHz) spectra were recorded on a Varian Inova spectrometer (Varian, USA). The signals of solvent residues were used as reference and were set at 4.79 for D2O and 7.26 for CCl3 The polyplexes of Dex-SSBAP-Dex at different nitrogen/phosphate (N/P) ratios were prepared by adding the polymer solution (800 L, different concentrations in 20 mM HEPES buffer at pH 7.4) to DNA (200 L of 75 g/mL in 20 mM HEPES buffer at pH 7.4), followed by vortexing for 5 s and incubating at room temperature for 30 min. Particle sizes and surface charges of the polyplexes were measured at 25 ºC with Nanosizer NS90 (Malvern, UK). To evaluate colloidal stability, saline solution was added to give a final salt concentration of 150 mM. The resulting polyplex solution was incubated at different time (0.5 and 4 h). The size of the polyplexes was measured by Nanosizer NS90. 2.4 Gel electrophoresis The polyplexes at different N/P ratios were made by adding 10 L of the polymer solution (varying concentrations in 20 mM HEPES at pH 7.4) to 10 L of DNA solution (80 g/mL in 20 mM HEPES), followed by vortexing for 5 s and the dispersions were incubated for 30 min at room temperature. Next, 10 L of HEPES buffer (as control) or the buffer containing DTT was added to set final DDT concentration of 10 mM, and the mixtures were incubated for another 30 min. After adding 3 L of 6× loading buffer (Fermentas), 10 L of this mixture was applied onto a 0.7% agarose gel with ethidium bromide (0.5 g/mL). After development of the gel, DNA was visualized using Tanon Gel Image system. 2.5 Cell culture and gene transfection in vitro MCF-7 and SKOV-3 cells (ATCC) were cultured in DMEM and McCoy's 5 medium, respectively, containing 10% FBS and 100 U/mL penicillin and streptomycin. Transfection experiments were performed using pCMV-GFP or pCMV-Luc reporter plasmid. The cells were plated in a 24-well plate (57×104 cells/well) and cultured in 0.5 mL of completed medium for at least 24 h until 60-70% cell confluence was reached. They were then washed twice with fresh 1×PBS buffer and incubated in the medium with or without 10% FBS for gene transfection. Next, the polyplexes at varying N/P ratios were prepared. In a typical transfection experiment of 24-well plate, the cells were treated with the polyplexes (1 g DNA) for 1 h at 37 °C in a 5% CO2-containing atmosphere. Then, the medium was replaced with completed medium and the cells were further incubated for another 47 h. A transfection formulation with B-pEI or L-pEI, prepared at an optimal N/P ratio of 8/1, was used as positive controls. All the transfection experiments were done in triplicates. GFP expression in transfected cells was determined by measuring GFP fluorescence intensity. In brief, the cells were washed twice with 1×PBS buffer and incubated with cell lysis buffer (200 L) at 4 ºC for 20 min. The cell lysate was collected and centrifuged to pellet cellular debris, and then 100 L of cell lysate was transferred in a 96-
C. Lin et al. / Dextranation of bioreducible cationic polyamide for systemic gene delivery
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well plate to test fluorescence intensity of GFP with excitation and emission wavelengths of 488 and 520 nm, respectively (Thermo Scientific, USA). Background fluorescence or auto-fluorescence was also measured with untreated cells as a blank control. Total protein concentration in collected cell lysate from each well was estimated with BAC Protein Assay kit (Invitrogen) and normalized using a standard BSA curve. GFP expression was presented as GFP fluorescence intensity (FI) normalized to protein mass and shown as arbitrary units (a. u.)/mg protein. To determine luciferase gene expression, luciferase activity was analyzed using a standard luciferase assay kit (Promega) with a luminometer (Thermo Scientific, USA). Luciferase expression was shown as relative light unit (RLU)/mg protein. All data were given as mean values (standard deviations) of three experiments. 2.6 Cell viability assay MCF-7 and SKOV-3 cells (5 × 103 cells/well) were respectively seeded in a 96-well plate. When the confluence reached 70~80%, the medium was replaced with serum-free medium. The cells were then exposed in Dex-SSBAP-Dex at polymer concentrations in the range from 2 to 100 μg/mL. After 1 h incubation, the cells were cultured in completed medium for another 47 h. Cell viability was determined by Alamar Blue assay. In brief, the cells were washed with fresh PBS and 200 L of 1× Alamar Blue-RMPI 1640 medium was added to each well. After 4 h of incubation, 100 L of the medium in each well was transferred to a 96-well plate for reading optical density (OD) at emission wavelengths of 570 nm and 630 nm, respectively, using Multiscan MK3 plate reader (Thermo Scientific, USA). Cell viability was calculated by the equation: Cell viability (%)= (ODsample –OD0)/ODcontrol – OD0)×100, where ODsample, ODcontrol, and OD0 are the OD values of the medium of treated cells, the medium of untreated cells, and 1×AlamarBlue-RMPI 1640 medium, respectively. The value for untreated cells (as control) was taken as 100% cell viability. 2.7 In vivo gene transfection in nude mice Animal experiments were officially approved by the Institutional Animal Care and Use Committee of Tongji University. The 4-6 week male Balb/c nude mice were intravenously administrated via tail vein with 400 l of polyplexes of Dex-SSBAP-Dex containing 50 g of pCMV-Luc and 5% glucose at an N/P ratio of 10/1. After 24 h, the mice were sacrificed and their liver, lung, spleens and kidneys were dissected, washed with PBS and homogenized in passive lysis buffer prepared by mixing 1 mL of 5× passive lysis buffer (Promega), 200 l of 50 mM phenylmethylslfonyl fluoride (PMSF) in methanol and 100 l of protease inhibitor (Sigma). The homogenate was centrifuged at 12,000 × g for 10 min at 4 °C. Then, 10 l of the supernatant was mixed with 100 μl of luciferase assay reagent (Promega) to determine luciferase expression. Data were given as mean values (standard deviations) of five experiments and expressed as relative light unit (RLU)/g tissue. 2.8 Statistical analysis Comparisons between two samples were performed with the student’s t-test. Differences were considered to be statistically significant at P
