Multifunctional Disulfide-Based Cationic Dextran Conjugates for

Jun 3, 2014 - ... delivery in vivo. Disulfide-based cationic dextran system thus has a high potential for intravenous gene delivery toward cancer gene...
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Multifunctional Disulfide-Based Cationic Dextran Conjugates for Intravenous Gene Delivery Targeting Ovarian Cancer Cells Yanyan Song,†,‡,§ Bo Lou,†,‡,§ Peng Zhao,† and Chao Lin*,† †

The Institute for Biomedical Engineering and Nanoscience, Tongji University School of Medicine, Tongji University, Shanghai 200092, P. R. China ‡ School of Life Sciences and Technology, Tongji University, Shanghai 200092, P. R. China S Supporting Information *

ABSTRACT: A folate-decorated, disulfide-based cationic dextran conjugate having dextran as the main chain and disulfidelinked 1,4-bis(3-aminopropyl)piperazine (BAP) residues as the grafts was designed and successfully prepared as a multifunctional gene delivery vector for targeted gene delivery to ovarian cancer SKOV-3 cells in vitro and in vivo. Initially, a new bioreducible cationic polyamide (denoted as pSSBAP) was prepared by polycondensation reaction of bis(p-nitrophenyl)-3,3′dithiodipropanoate, a disulfide-containing monomer, and BAP. It was found that the pSSBAP was highly efficient for in vitro gene delivery against MCF-7 and SKOV-3 cell lines. Subsequently, two cationic dextran conjugates with different amounts of BAP residues (denoted as Dex-SSBAP6 and Dex-SSBAP30, respectively) were synthesized by coupling BAP to disulfide-linked carboxylated dextran or coupling pSSBAP-oligomer to p-nitrophenyl carbonated dextran. Both two conjugates were able to bind DNA to form nanosized polyplexes with an improved colloidal stability in physiological conditions. The polyplexes, however, were rapidly dissociated to liberate DNA in a reducing environment. In vitro transfection experiments revealed that the polyplexes of Dex-SSBAP30 efficiently transfected SKOV-3 cells, yielding transfection efficiency that is comparable to that of linear polyethylenimine or lipofectamine 2000. AlamarBlue assay showed that the conjugates had low cytotoxicity in vitro at a high concentration of 100 mg/L. Further, Dex-SSBAP30 has primary amine side groups and thus allows for folate (FA) conjugation, yielding FA-coupled Dex-SSBAP30 (Dex-SSBAP30-FA). It was found that Dex-SSBAP30-FA was efficient for targeted gene delivery to SKOV-3 tumor xenografted in a nude mouse model by intravenous injection, inducing a higher level of gene expression in the tumor as compared to Dex-SSBAP30 lacking FA and comparable gene expression to linear polyethylenimine as one of the most efficient polymeric vectors for intravenous gene delivery in vivo. Disulfide-based cationic dextran system thus has a high potential for intravenous gene delivery toward cancer gene therapy. KEYWORDS: disulfide, dextran, systemic gene delivery, targeting, ovarian cancer cells

1. INTRODUCTION The availability of safe and highly efficient vectors for systemic gene delivery remains a big challenge for the success of gene therapy.1 Although recombinant viral vectors such as adenoassociated virus have been widely employed for in vitro and in vivo gene transfer due to their high transfection ability, further clinical utilization of these viral vectors is seriously impeded by their controversial safety issues such as immunogenicity and oncogenicity.2 Synthetic cationic polymers as nonviral vectors offer new opportunity for relatively safe gene delivery because they regularly induce a low immune response and have © XXXX American Chemical Society

advantages such as high gene-loading capacity, handy chemical modification, and large-scale production.3 For these reasons, in the past two decades synthetic cationic polymers such as polyethylenimine (pEI) and polylysine have been investigated widely for nonviral gene delivery.4,5 It is known that cationic polymers like pEI condense DNA into nanoscale cationic Received: November 6, 2013 Revised: May 14, 2014 Accepted: June 3, 2014

A

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which in turn causes facilitated gene release, giving rise to high transfection efficiency.17 Although a number of bioreducible cationic polymers were highly efficient for nonviral gene delivery in vitro,17−19 their further application for gene delivery in vivo was reported in few literatures. For example, Kim et al. indicated that bioreducible poly(oligo-D-arginine) induced a high level of heme oxygenase-1 expression in mouse striatum by local stereotaxic injection.20 We showed that disulfide-based pEI/hTERT-siRNA nanocomplexes efficiently inhibited HepG2 tumor growth by the injection of the nanocomplexes in the tumor bearing in a nude mouse model.21 Although these bioreducible polymeric systems are suited for local gene delivery, they are not ideal candidates for systemic gene delivery because their polyplexes lack colloidal stability and targeting ability. To address these problems, we prepared PEGylated bioreducible poly(amido amine)s, which condensed DNA into PEGylated polyplexes with an improved colloidal stability under physiological conditions.22 In another study, Lee and co-workers showed RVG-tethered-PEGylated bioreducible pEIs for systemic gene delivery targeting brain in a mouse model.23 However, to the best of our knowledge, no report has appeared on dextranated bioreducible cationic polymers for systemic gene delivery. In this study, we aim to design a disulfide-based cationic dextran system for i.v. gene delivery targeting tumor. Initially, a new bioreducible cationic polyamide with primary amine ending groups was synthesized by stepwise polycondensation reaction of bis(p-nitrophenyl)-3,3′-dithiodipropanoate and 1,4bis(3-aminopropyl)piperazine (BAP), thus being denoted as pSSBAP (Scheme 1). The possibility of pSSBAP for nonviral

polyplexes, which can mediate a facilitated endosomal escape due to a high buffering capacity (proton sponge effect) and protect gene from degradation by nuclease, affording detectable transfection efficiency in vitro.6 However, systemic gene delivery of cationic polyplexes is hampered by inherent extracellular barriers.7,8 For example, after intravenous (i.v.) injection of cationic polyplexes, their nonspecific interaction with opsonins in the blood is a major obstacle to systemic gene delivery. As such, opsonized polyplexes tend to be rapidly eliminated by the phagocytic system such as liver and spleen.9 Besides, the salts at a high ionic strength may weaken electrostatic interactions between cationic polymers and DNA. This unfavorable process makes cationic polyplexes colloidally unstable in physiological conditions, and as a result, the aggregates are regularly formed, which are rather inefficient for gene transfer. Accordingly, most of current polymeric vectors are not ideal for systemic gene delivery, although efficient for in vitro gene delivery. In order to design polymeric vectors for systemic gene delivery, one typical approach is a chemical modification of cationic polymers with poly(ethylene glycol) (PEG), a nontoxic neutral biomaterial. This PEGylation method offers PEGylated cationic polymers, which can condense DNA into PEGylated polyplexes with near-neutral PEG surface. It is indicated that, compared to native cationic polyplexes, PEGylated polyplexes regularly display an improved colloidal stability in physiological conditions and minimized interactions with serum opsonins, leading to prolonged circulation and lower toxicity.10,11 These properties make PEGylated polyplexes more efficient for systemic gene delivery. As an alternative method to PEGylation, dextranation of cationic polymers is also applicable to decorate cationic polymers. Similar to PEG, dextran is also a nontoxic, neutral biomaterial. Cheng et al. synthesized 10 kDa dextranconjugated branched pEIs, and they indicated that polyplexes of dextran-grafted pEIs revealed an improved colloidal stability in physiological conditions, implying the possibility for systemic gene delivery.12 Furthermore, different from linear PEG with two terminal groups, linear dextran has a lot of hydroxyl side groups amenable to different chemical modifications. Thus, cationic dextran conjugates based on different polycations such as low molecular weight pEI and oligoamines were investigated as nonviral vectors for gene delivery.13,14 As a typical example, Domb et al. showed that dextran−spermine conjugate was efficient for systemic gene delivery in a mouse model by intranasal or intramuscular administration.15 However, they also found that this conjugate was inefficient for i.v. gene delivery and that detectable in vivo transfection efficiency was only obtained using PEGylated dextran−spermine.15 Besides, other cationic dextran systems were not reported for systemic gene delivery. This is likely because these current cationic dextran systems are not finely designed to endow their polyplexes with colloidal stability, controlled gene release, and targeting ability, which are favorable properties for systemic gene delivery. In recent years, there is an increasing interest in disulfidebased (bioreducible) cationic polymers designed for nonviral gene delivery.16 It has been found that the concentration of the glutathione is about 1000 times higher in the intracellular environment compared to the extracellular environment. Hence, disulfide bonds are chemically stable extracellularly, but redox-sensitive in an intracellular reducing environment. By intracellular cleavage of disulfide bonds in bioreducible cationic polymers, their polyplexes can undergo an adequate unpacking

Scheme 1. Synthesis of Bioreducible Cationic Polyamide (pSSBAP)

gene delivery was evaluated in vitro against SKOV-3 and MCF7 cells. Subsequently, two cationic dextran conjugates were prepared, which have linear dextran as the main chain and different amounts of disulfide-linked BAP residues as the grafts (denoted as Dex-SSBAP6 and Dex-SSBAP30, respectively, Scheme 2). Gene delivery properties of the conjugates were investigated in terms of buffering capacity, gene binding and release behaviors, and polyplex colloidal stability in physiological conditions. In vitro transfection activity and cytotoxicity of the conjugates were assessed in SKOV-3 cells. Finally, folate, a high-affinity ligand for folate receptor, was coupled to DexSSBAP30 through carbodiimide chemistry, affording folatecoupled Dex-SSBAP30 (denoted as Dex-SSBAP30-FA, Scheme 3) for targeted gene delivery to SKOV-3 cells overexpressing folate receptor.24,25 Systemic gene delivery was conducted by B

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Scheme 2. Synthesis of Disulfide-Based Cationic Dextran Conjugates (a) Dex-SSBAP6 and (b) Dex-SSBAP30a

a

The DS of Dex-PNC and Dex-SH were 20, that is, about 6 of PNC or SH groups per dextran chain.

dimethyl sulfoxide (DMSO), dithiothreitol (DTT), p-nitrophenol, and branched polyethylenimine (B-pEI, Mw = 25k Da) were ordered from Sigma-Aldrich. Linear polyethylenimine (LpEI, Mw = 22 kDa) was ordered from Polysciences, Inc. (USA). The plasmids, pCMV-GFP, pCMV-Luc, and pCMV-LacZ, encoding GFP, Luc, and LacZ gene, respectively, under the control of a CMV promoter, were ordered from Plasmid Factory (Germany). Dextran derivatives, p-nitrophenyl carbonated dextran (Dex-PNC), and thiolated dextran (Dex-SH) at a substitution degree (DS, defined as the number of PNC/SH group per 100 anhydroglucose unit in dextran) of 20, that is, ∼6 reactive group per dextran chain, were prepared according to our previous work.26 2.2. Synthesis of Monomers. 2.2.1. Synthesis of Bis(pnitrophenyl)-3,3′-dithiodipropanoate (DTPA-PNC). Bis(p-nitrophenyl)-3,3′-dithiodipropionate (DTPA-PNC) was obtained by the method in ref 27. As a typical experiment, 3,3′dithiodipropionic acid (5 g, 24 mmol) was reacted with an excess thionyl chloride (20 mL) in a three-neck round-bottom flask (150 mL) at 85 °C and refluxed for 4 h. After the removal of unreacted thionyl chloride through rotary evaporation, dithiodipropionic chloride was obtained as a yellowish liquid and used without further purification. Then, dithiodipropionic chloride (5.9 g, 24 mmol) in anhydrous acetone (50 mL) was dropwise added into a mixture of p-nitrophenol (6.7 g, 48 mmol) and pyridine (3.8 g, 48 mmol) in cooled acetone (100 mL) for 2 h at 0 °C, and the mixture was stirred at room temperature overnight. The resulting mixture was finally poured in deionized water (800 mL) to give a precipitate. DTPA-PNC was obtained as an acicular crystal by recrystallization in ethyl acetate (25 mL) (yield: 2.4 g). 1H NMR (500 MHz, CCl3): δ 3.1 (s, 8H, 2 × CH2CH2S); 7.3 and 8.3 (aromatic protons). Electron ionization mass spectrum

Scheme 3. Synthesis of Folate-Coupled Dex-SSBAP30 (DexSSBAP30-FA)

i.v. injection of polyplexes of Dex-SSBAP30 or Dex-SSBAP30FA in SKOV-3 tumor-xenografted nude mice using the plasmid DNA encoding luciferase or LacZ gene. Biodistribution of transgene expression in organs and tumor was evaluated by luciferase expression assay or X-Gal staining of betagalactosidase. Besides, circulation kinetics and biodistribution of Dex-SSBAP30-FA-based polyplexes were investigated in a mouse model.

2. MATERIALS AND METHODS 2.1. Materials. 3,3′-Dithiodipropionic acid (DTPA) and 1,4-bis(3-aminopropyl)piperazine (BAP), 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were ordered from AlfaAesar. Dextran (Mw = 5k Da), pyridine, dithiothreitol (DTT), p-nitrophenyl chloroformate (PNC), anhydrous dimethylformamide (DMF), C

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(LCMS-2020, Shimadzu (Hong Kong), Ltd.) shows m/z = 452 (M+) and C18H17N2O8S2 requires M+ = 452.0 (Figure S1, Supporting Information). 2.2.2. Synthesis of 2-Carboxyethyl 2-Pyridyl Disulfide (PDP). 2-Carboxyethyl 2-pyridyl disulfide (PDP) was synthesized according to the literature.28 Briefly, 3-mercaptopropionic acid (4.5 g) was reacted with 2,2′-dipyridyldisulfide (18.8 g) in anhydrous ethanol (75 mL). After the removal of the ethanol by rotary evaporation, crude product was purified on a silica column with petroleum ether/ethyl acetate (3/2, v/v) to give a waxy solid (yield: 4.5 g). 1H NMR (500 MHz, DMSO): δ 2.6 (t, 2H, CH2CH2COOH); 3.0 (t, 2H, CH2CH2COOH); 7.25 7.75, 7.83, and 8.46 (protons in pyridyl ring) (Figure S2, Supporting Information). 2.3. Synthesis of Disulfide-Based Polymers. 2.3.1. Synthesis of Primary Amine-Terminated Bioreducible Cationic Polyamide (pSSBAP). pSSBAP was synthesized by stepwise polycondensation reaction of bis(p-nitrophenyl)-3,3′-dithiodipropionate (DTPA-PNC) and 1,4-bis(3-aminopropyl)piperazine (BAP) (Scheme 1). In a typical experimental procedure, DTPA-PNC (0.5 g, 1.1 mmol) was reacted with BAP (0.22 g, 1.1 mmol) in a round-bottom brown flask and dissolved in anhydrous DMSO (2 mL) as a solvent. The reaction proceeded at 40 °C under nitrogen protection for 5 days. Next, 10 mol % excess of BAP (0.02 g) was added into the system for another 2 days. Finally, the mixture was diluted with ∼30 mL of deionized water, acidified to pH ≈ 5 with 6 M HCl, and then purified by ultrafiltration operation (1000 MWCO). The pSSBAP was obtained as its HCl salt after freeze-drying (yield: 0.34 g). 1H NMR (500 MHz, D2O): δ (ppm) = 1.95 (4H, 2 × N−CH2CH2CH2); 2.66 (4H, 2 × N− CH2CH2CH2); 2.92 (4H, 2 × N−CH2CH2CH2); 3.28 (8H, CH2CH2SSCH2CH2); 3.3−4.1 (8H, 2 × N−CH2CH2−N) (Figure S3, Supporting Information). 2.3.2. Synthesis of Disulfide-Based Cationic Dextran Conjugates. Dex-SSBAP6 was prepared by a two-step procedure (Scheme 2). First, thiolated dextran (Dex-SH) at a substitution degree (DS) of 20 (200 mg, 0.24 mmol SH) and PDP (103 mg, 0.48 mmol) were dissolved in 4 mL of anhydrous DMF and stirred under nitrogen atmosphere for 24 h at room temperature. Carboxylated dextran (Dex-SS-COOH) was finally obtained as a white powder by ultrafiltration purification (MWCO 1000) and freeze-drying (yield: 150 mg). Next, Dex-SS-COOH (100 mg, 0.12 mmol COOH groups) was activated with EDC·HCl (70 mg, 0.36 mmol) and NHS (42 mg, 0.36 mmol) in MES buffer (5 mL, pH 6.5) for 15 min at 0 °C. The mixture was then dropwise added into BAP (240 mg, 1.2 mmol) in the MES buffer (1 mL) at 0 °C, and the reaction proceeded for 48 h at room temperature. The resulting product Dex-SSBAP6 was obtained by ultrafiltration purification (MWCO 1000) with NaCl solution (150 mM) and deionized water as well as freeze-drying (yield: 120 mg). The composition of Dex-SSBAP was determined by 1H NMR (Figure S4a, Supporting Information). Dex-SSBAP30 was prepared by a two-step procedure (Scheme 2). In a typical synthesis protocol, SSBAP-oligomer with an average-number polymerization degree of 5 was prepared by reacting DTPA-PNC (344 mg) with an excess of BAP (253 mg, stoichiometric ratio (r) of DTPA-PNC/BAP is 0.67) in DMSO (1 mL) in a brown flask under nitrogen atmosphere at 40 °C for 3 days. Dex-PNC (DS 20, 50 mg) in DMSO (1 mL) was then dropwise added into the reaction system within 1 h, and the reaction was continued for another

48 h under nitrogen atmosphere at room temperature. The resulting solution was diluted with deionized water to ∼30 mL and acidified to pH ≈ 5 with HCl (4 M). Dex-SSBAP30 was obtained in HCl salt form as a solid powder after ultrafiltration purification (MWCO 5000) and freeze-drying (yield: 55 mg). The composition of Dex-SSBAP30 was determined by 1H NMR and shown in Figure S4b, Supporting Information. 2.3.3. Synthesis of Folate-Coupled Dex-SSBAP30. Folatecoupled Dex-SSBAP30 was prepared by coupling of folate to Dex-SSBAP30 via DCC/NHS activation (Scheme 3). In brief, folate (3.5 mg) was activated with EDC (1.5 mg) and NHS (1 mg) in DMSO (1 mL) for 1 h at room temperature, after which was added Dex-SSBAP30 (30 mg) in DMSO (1 mL) and pyridine (50 μL). The mixture was then diluted with deionized water (1 mL) and stirred overnight at room temperature. The solution was filtered through a filter (0.45 μm) and purified by ultrafiltration (MWCO 1000). Dex-SSBAP30-FA was obtained as a yellowish powder (yield: 28 mg) after freeze-drying. The composition of Dex-SSBAP30-FA was determined by 1H NMR (Figure 4). 2.3.4. Synthesis of Cy5.5-Labeled Dex-SSBAP30, DexSSBAP30-FA, and L-pEI. The polymers were labeled with Cy5.5 by coupling of Cy5.5-NHS to an amine group in the polymers. In an experiment for the preparation of Cy5.5labeled Dex-SSBAP30-FA, 50 mg of Dex-SSBAP30-FA in DMSO (1 mL) was mixed with Cy5.5-NHS (20 μL DMSO, 10 nmol/μL) for coupling overnight. Cy5.5-labeled DexSSBAP30-FA was collected as a powder in blue after ultrafiltration (1000 g/mol cutoff) and freeze-drying. The amount of Cy5.5 in Dex-SSBAP30-FA was calculated to be 140 pmol/μg by using a calibration curve from a series of Cy5.5 standard solutions at different concentrations. The amount of Cy5.5 in Dex-SSBAP30 and L-pEI was 143 and 52 pmol/μg, respectively. 2.4. Chemical and Biophysical Characterizations. 2.4.1. Characterization. Chemical composition of monomers and polymers was characterized by 1H NMR analysis on Varian Inova spectrometer (500 MHz, Varian). Molecular weight and polydispersity of pSSBAP were measured by gel permeation chromatography (Viscoteck GPCmax Instrument) equipped with A6000 M general mixed aqueous column (300 × 7.8) and Triple Detection System (Malvern, U.K.), using 0.3 M NaAc containing 30% ethanol as an elute at a flow rate of 1 mL/min. The sample was prepared at a concentration of 4 mg/mL in the eluent and 100 μL was injected. The buffering capacity of pSSBAP was determined by acid−base titration according to our previous report.29 2.4.2. Particle Size and Zeta-Potential Measurements. The polyplexes were prepared at different nitrogen/phosphate (N/ P) ratios (5, 10, and 20, respectively) by mixing polymer solution at different concentrations (800 μL in 20 mM HEPES buffer, pH 7.4) with DNA solution (200 μL, 75 μg/mL in 20 mM HEPES buffer, pH 7.4) and subsequent standing at room temperature for 30 min. Particle size and surface charge of polyplexes were tested with Nanosizer NS90 (Malvern, U.K.) at 25 °C. 2.4.3. Agarose Gel Retardation. The polyplexes at different N/P ratios were prepared by adding different concentrations of polymer solution (10 μL, 20 mM HEPES, pH 7.4) to DNA solution (10 μL, 80 μg/mL in 20 mM HEPES, pH 7.4), followed by vortexing for 5 s, and the dispersions were incubated for 30 min at room temperature. Next, HEPES buffer (10 μL, as control) or the buffer containing DTT was incubated D

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with the polyplex solution for 30 min at a final DTT concentration of 10 mM. After adding 6 × loading buffer (3 μL, Fermentas), the mixture (10 μL) was loaded on a 0.7% agarose gel with ethidium bromide (0.5 μg/mL). DNA was visualized and captured using Tanon Gel Image system. 2.5. In Vitro Transfection Experiment, Transfection Efficiency, and Cell Viability Assay. 2.5.1. Transfection Protocol. MCF-7 and SKOV-3 cells (ATCC, USA) were cultured in RPMI 1640 and Mccoy’s-5α, respectively, containing 10% FBS and 100 U/mL penicillin/streptomycin (GIBCO) at 37 °C in a humidified 5% CO2. In vitro transfection experiment was performed using pCMV-GFP or pCMV-Luc reporter plasmid. First, the cells (7 × 104 cells/ well) were plated in a 24-well plate and cultured in complete culture medium (0.5 mL) for at least 24 h before the cell confluence was 60−70%. The cells were then washed twice with fresh 1 × PBS buffer and incubated in the medium with or without 10% FBS for subsequent transfection. Next, the polyplexes at different N/P ratios were prepared by adding polymer solution (100 μL, varying concentrations in 20 mM HEPES buffer, pH 7.4) to DNA solution (50 μL, 20 mM HEPES buffer, pH 7.4), followed by gentle shaking and incubation at room temperature for 30 min. In a typical transfection test, the cells were coincubated with the polyplexes (1 μg DNA) for 1, and the medium was then replaced with fresh complete culture medium (0.5 mL). After 47 h posttransfection, the cells were used for assay. A transfection formulation with B-pEI or L-pEI, prepared at an optimal N/P ratio of 8/1, or with Lipofectamine 2000 (1.5 μL+1 μg DNA), were used as positive controls. All the cell experiments were tested in triplicates. 2.5.2. Transfection Efficiency Assay. Transfection efficiency was determined using fluorescence plate reader (Thermo Fisher Scientific, USA) by the quantification of fluorescence intensity of GFP or luciferase expressed in transfected cells. Briefly, after transfection, the cells in a 24-well plate were washed twice with 1× PBS buffer and incubated with cell lysis buffer (200 μL) at 4 °C for 20 min. The cell lysates were collected and centrifuged to pellet cellular debris, and 100 μL of cell lysate was transferred in a 96-well plate. Fluorescence intensity of GFP was determined with fluorescence plate reader (Thermo Fisher Scientific, USA) with the excitation and emission wavelengths at 488 and 520 nm, respectively. Background fluorescence was also determined with untreated cells as a blank (control). The recovered protein concentration in cell lysate was determined with a BAC protein assay kit (Invitrogen). Transfection efficiency was calculated as the fluorescence intensity (FI) normalized to protein mass and presented as arbitrary units (a.u.)/mg protein. Luciferase expression was quantified using a luciferase assay system (#E1500, promega) and recorded with luciferase plate reader (Thermo Fisher Scientific, USA). Transfection efficiency was presented as relative light unit (RLU) relative to protein mass and given as RLU/mg protein. The results are given as mean ± standard deviation for triplicate samples. 2.5.3. Cytotoxicity Test. The cytotoxicity of as-prepared polymers was assessed by the assay of cell viability. In brief, MCF-7 or SKOV-3 cells (1 × 104 cells/well in a 96-well plate) were coincubated with the polymers at varying concentrations (1−100 mg/L) in the culture medium without FBS for 1 h. The cells were washed twice with 1× PBS and incubated for another 47 h in complete culture medium. Cell metabolic

activity was detected using AlamarBlue assay (Invitrogen). The value for untreated cells was taken as 100% cell viability. 2.6. Cellular Uptake Study in SKOV-3 Cells. For cellular uptake study, pCMV-LacZ plasmid was labeled with nucleic acid staining YOYO-1 iodide (Molecular Probes). The plasmid (10 μg) was incubated with YOYO-1 (10 μL, 0.01 mM) for 2 h in the dark. Labeled pDNA was used to prepare polyplexes for cellular uptake study. In a typical procedure, SKOV-3 cells (1.5 × 105 cells/well) were seeded in a 6-well plate and cultured in complete culture medium (2 mL) for at least 24 h until 60− 70% cell confluence was reached. Next, the cells were washed twice with 1× PBS and incubated with the polyplexes (containing 2 μg of pDNA), prepared at an optimal N/P ratio, in complete culture medium with or without folate (100− 400 μM) for 4 h. The cells were washed twice with ice-cold 1× PBS. To quench the fluorescence of polyplexes adsorbed on the cellular surface, the cells were treated with 0.4% trypan bluecontaining PBS buffer for 5 min. After washed with 1× PBS and trypsinized, the cells were harvested and resuspended in 1× PBS. Cellular uptake was evaluated by a flow cytometry (BD) at 10,000 cells gated per sample. 2.7. Transgene Expression by Tail-Vein Injection of Polyplexes in Nude Mice. All animal experiments were officially approved by the Institutional Animal Care and Use Committee of Tongji University (Shanghai, P. R. China). A xenograft SKOV-3 tumor model was prepared by subcutaneous injection of SKOV-3 cells (5 × 106 cells/tumor) into the flank region of six-week male nude mice (Balb/c) (SLAC, Shanghai, P. R. China). As the tumor reached about 200−300 mm3 (∼0.15−0.2 g), polyplex formulation of Dex-SSBAP30 or DexSSBAP30-FA, prepared at an optimal N/P ratio of 10/1, containing 50 μg of pCMV-Luc in 20 mM HEPES buffer (400 μL, 5% glucose, pH 7.4), was injected by tail vein into nude mice (n = 5). The polyplexes of L-pEI at an N/P ratio of 8/1 were also used as a positive control. After 24 h injection, the mice were sacrificed. The tissues, i.e., liver, lung, spleen, and kidney, and tumor were harvested, washed with 1× PBS, weighted, and homogenized (Mini-beadbeater-1, Biospec, USA) in a tissue lysis buffer prepared with 5× passive lysis buffer (1 mL, #E1941, Promega), 50 mM phenylmethylslfonyl fluoride in methanol (200 μL, #P7626, Sigma), and protease inhibitor (100 μL, #P8340, Sigma). The homogenate was centrifuged at 12,000g and 4 °C for 10 min, and the supernatant (100 μL) was mixed with luciferase assay reagent (100 μL, #E1050, Promega). Luciferase expression in each tissue was determined with luciferase plate reader (Thermo Fisher Scientific, USA) and presented as relative light unit (RLU)/g tissue. The polyplexes of Dex-SSBAP30-FA containing 50 μg of pCMV-LacZ were also used for i.v. injection in the same nude mouse model. After 24 h post-i.v. injection, the mice were sacrificed. The tissues were immersed in 4% paraformaldehyde (PFA) at 4 °C for 48 h. Next, the PFA was replaced with different concentrations of sucrose solution in 1× PBS (10%, 20%, and 30%). After these tissue blocks were frozen in O.C.T. compound, tissue section (10 μm) was cut with cryostat microtome (Leica, CM1510) and laid on a microscope slide. The section was finally stained using X-Gal kit according to the manufacturer’s instruction (Invitrogen). The LacZ expression as pale blue was captured with an optical microscope (Nikon). 2.8. Biodistribution of Polyplexes of Cy5.5-Labeled Polymers in Nude Mice. Six week-old male BALB/c nude mice were ordered from Shanghai Laboratory Animal Center E

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Figure 1. (a) Particle size (bar) and zeta-potential (line) of pSSBAP-based polyplexes at N/P ratios from 10/1 to 30/1; (b) agarose gel retardation analysis of pSSBAP-based polyplexes at N/P ratios in the absence (w/o) or presence (w/) of dithiothreitol (DTT, 5 mM); (c) transfection efficiency of pSSBAP as a function of N/P ratios against MCF-7 and SKOV-3 cells, pSSBAP vs. B-pEI: *P < 0.05, **P < 0.01, ***P < 0.001; (d) Cytotoxicity of pSSBAP as a function of polymer concentrations from 1 to 100 mg/L against MCF-7 and SKOV-3 cells.

(Shanghai, P. R. China). SKOV-3 tumors were induced in the mice by subcutaneously injecting SKOV-3 cells (5 × 106) into the flank of the mice. When the tumor has reached to about 300−500 mm3, the polyplexes of Cy5.5-labeled polymers (DexSSBAP30-FA or Dex-SSBAP30), formed at an N/P of 10/1, in HEPES buffer (200 μL, pH 7.4) was i.v. administrated via tailvein injection. The amount of Cy5.5 of the polyplexes is 4200 pmol, and DNA dose is 30 μg. Then, at regular time intervals, 3D tomographic scanning was run with the FMT 2500 system (PerkinElmer) using excitation and emission wavelengths of 670 and 690−740 nm, respectively. The scan data were analyzed with a reconstruction software (True Quant 3D, v.2.0.0.19) offered by the manufacturer (PerkinElmer). Finally, the organs (liver, kidneys, spleen, and lungs) and tumor were isolated and 2D-ex vivo fluorescence scanning was captured. For data analysis, the region of tumor was selected and the amount (pmol) of Cy5.5 was quantified with the software according to the manufacturer’s instructions. The accumulation (%ID) of polyplexes in tumor is calculated as the amount of Cy5.5 in tumor compared to the total injected Cy5.5 dose (4200 pmol). The data are given as the mean ± SD in triple tests. 2.9. Pharmacokinetic Analysis of Polymers in Mice. Four week-old male/female Kunming mice (20−25 g) were ordered from Shanghai Laboratory Animal Center (Shanghai, P. R. China). Circulation kinetics of Dex-SSBAP30-FA or L-pEI in mice was evaluated with Cy5.5-labeled polymer according to the method by Huang et al.30 In brief, the polyplexes of Cy5.5labeled polymers (Dex-SSBAP30-FA or L-pEI), formed at an N/P of 10/1, in HEPES buffer (200 μL, pH 7.4) were used for tail-vein injection (3 male/3 female mice per group). The Cy5.5 dose in polyplexes is 4200 pmol, and DNA dose is 30 μg. Then, at different time intervals, 100 μL of blood samples were collected and plasma was then obtained after centrifugation at 1000 rpm. The amount (pmol) of Cy5.5 in serum sample was quantified with FMT 2500 system (PerkinElmer). The accumulation (%ID) of the polymer in plasma was calculated as the percentage of Cy5.5 in plasma compared to the total injected Cy5.5 dose (4200 pmol). A similar experiment was

also performed with polyplexes of Cy5.5-labeled L-pEI as a control. The data are given as the mean ± SD in 4−6 tests. 2.10. Statistical Analysis. Statistical analysis was performed using the student’s t test. A difference was considered to be statistically significant when P < 0.05.

3. RESULTS AND DISCUSSION 3.1. Design of a New Bioreducible Cationic Polyamide for Gene Delivery in Vitro. In this study, a new bioreducible cationic polyamide with primary amine terminal group was synthesized by polycondensation reaction of bis(p-nitrophenyl)-3,3′-dithiodipropanoate (DTPA-PNC) and 1,4-bis(3aminopropyl)piperazine (BAP) in anhydrous DMSO, thus being denoted as pSSBAP (Scheme 1). An equal molar ratio of DTPA-PNC and BAP was applied in the synthesis, in order to produce pSSBAP with the highest theoretical molecular weight. This reaction proceeded for 5 days, resulting in a yellowish viscous solution. To consume any unreacted p-nitrophenyl ester residue, 10% excess of BAP was then added in the reaction for another 2 days, which makes resulting pSSBAP have primary amine terminal groups. Finally, pSSBAP was obtained as its HCl-salt form after ultrafiltration (1000 g/mol cutoff) and freeze-drying. This polymer was well soluble in HEPES buffer (20 mM, pH 7.4) at a tested polymer concentration of 5 mg/ mL. 1H NMR spectra of pSSBAP were in good line with its chemical compositions (Figure S3a, Supporting Information). Besides, in the spectra, there is no signal at δ 7.3 and δ 8.3, attributed to the aromatic protons in p-nitrophenyl residue, suggesting that p-nitrophenyl ester residue is consumed completely. GPC analysis indicated that pSSBAP had weightaveraged molecular weight of 8.3 kg/mol with a polydispersity index (PDI) of 1.3 (Figure S3b, Supporting Information). The results suggest that a new bioreducible cationic polyamide can be generated through the polycondensation reaction between DTPA-PNC and tertiary amine-containing primary diamines like BAP. Gene delivery properties of pSSBAP were studied in terms of buffering capacity, particle size, surface charge as well as gene F

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Figure 2. Particle size (a) and zeta potential (b) of the polyplexes of Dex-SSBAP6 and Dex-SSBAP30 at different N/P ratios; (c) effect of NaCl (150 mM) on the particle size of the polyplexes, prepared at an N/P ratio of 20/1, after incubation of 0.5 and 4 h; (d) agarose gel electrophoresis analysis of the polyplexes of Dex-SSBAP6 and Dex-SSBAP30 at different N/P ratios without (w/o) or with (w/) dithiothreitol (DTT, 5 mM).

binding and release behavior. First, acid−base titration was carried out to evaluate the buffering capacity of pSSBAP (Figure S4, Supporting Information), defined as the percentage of protonable amine groups as they are protonated from pH 7.4 (extracellular environment) to pH 5.1 (endosomal environment). It was revealed that pSSBAP had higher buffering capacity (20.4%) as compared to 25 kDa pEI (15.6%), suggesting that pSSBAP may mediate facilitated endosomal escape. In the previous reports, poly(amido amine)s containing piperazine residues also displayed higher buffering capacity than pEI.31,32 Next, the particle size and surface charge of pSSBAPbased polyplexes were characterized by dynamic light scattering (DLS) analysis. As revealed in Figure 1a, pSSBAP condensed DNA into nanosized, positively charged polyplexes with particle sizes below 200 nm in diameter. Besides, when the N/P ratios were increased from 10/1 to 30/1, the particle sizes of the polyplexes reduced from 146 ± 4 to 112 ± 3 nm and their surface charges ranged from +23.2 ± 1.6 to +25.3 ± 1.2 mV. Agarose gel retardation analysis further revealed gene binding ability of pSSBAP (Figure 1b). When the N/P ratios were increased from 0.25/1 to 20/1, the mobility of DNA was completely retarded at and above an N/P ratio of 1/1, suggesting a complexation of pSSBAP and DNA. However, after incubating the polyplexes with dithiothreitol (DTT), mimicking an intracellular reducing environment, an adequate DNA release was observed. This implied that intracellular cleavage of the disulfide in pSSBAP may cause facilitated DNA release. These favorable gene delivery properties including nanoscale particle size, positive surface charge, high buffer capacity, and triggered gene release give a hint that pSSBAP may be effective for gene delivery in vitro. In vitro gene transfection of pSSBAP was performed against MCF-7 and SKOV-3 cells after the cells were transfected by the polyplexes of pSSBPA containing the plasmid DNA encoding green fluorescent protein (GFP) gene. The polyplexes can potently transfect MCF-7 cells, producing GFP expression (Figure S5, Supporting Information). Moreover, with increasing N/P ratios from 10/1 to 50/1, the transfection efficiencies of pSSBAP significantly increased (Figure 1c). Further, the efficiencies were comparable in SKOV-3 cells and about 3−8 times higher in MCF-7 cells as compared to those afforded by branched pEI (B-pEI) as one of the most efficient polymeric

gene carriers. Figure 1d exhibits the cytotoxicity of MCF-7 and SKOV-3 cells, as determined by AlamarBlue assay, after exposed in pSSBAP at different concentrations ranging from 1 to 100 mg/L. As expected, pSSBAP had very low cytotoxicity with at least 90% cells maintaining survival. The data again support the conclusion that bioreducible cationic polymers normally cause low cytotoxicity in vitro.17 Overall, a new bioreducible cationic polyamide (pSSBAP) is found as a nonviral vector for efficient gene delivery in vitro. 3.2. Design of Disulfide-Based Cationic Dextran Conjugates for Gene Delivery in Vitro. Disulfide-based cationic dextran conjugates containing different amounts of BAP residues (denoted as Dex-SSBAP) were prepared, which have dextran as the main chain and disulfide-linked BAP residue in the side chain. In the first route, disulfide-based carboxylated dextran (denoted as Dex-SS-COOH) at a substitution degree (DS) of 20 was synthesized by reacting thiolated dextran (DS 20) with 2-carboxyethyl 2-pyridyl disulfide (PDP). Subsequently, Dex-SS-COOH was conjugated with BAP by carbodiimide chemistry, yielding the conjugate with ∼6 BAP residues per dextran chain (denoted as DexSSBAP6, Scheme 2a). In another route, a pSSBAP-oligomer with primary amine terminal group was synthesized. The number-average polymerization degree (Xn) of the oligomer was controlled by using a stoichiometric ratio (r) of the monomers (i.e., rDTPA‑PNC/BAP = 0.67) and was calculated with the equation Xn = (1 + r)/(1 − r). Subsequently, p-nitrophenyl carbonated dextran at a DS of 20 was conjugated with pSSBAPoligomer (Xn=5), yielding the conjugate with ∼30 BAP residues per dextran chain (denoted as Dex-SSBAP30, Scheme 2b). In the synthesis process, ca. 10-folded molar excess of BAP or pSSBAP-oligomer relative to reactive group of dextran was used in the reaction to avoid the cross-linking of dextran chains. 1H NMR analysis of Dex-SSBAP6 and Dex-SSBAP30 showed that their spectra were in line with expected structures (Figure S6, Supporting Information). Moreover, by comparing the integrals of the signals at δ 1.8 (CH2CH2CH2 in BAP residue) and δ 4.95 (anomeric proton in dextran), the amounts of BAP residue in Dex-SSBAP6 and Dex-SSBAP30 can be calculated to be ∼6 and 30 per dextran chain, respectively. GPC analysis showed that Dex-SSBAP30 had weight-averaged molecular weight of 18.5 kg/mol (PDI = 1.67). The results indicate that DexG

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Figure 3. AlamarBlue assay showing cell viability of (a) SKOV-3 cells and (b) MCF-7 cells at different polymer concentrations; (c) transfection efficiency of Dex-SSBAP6 and Dex-SSBAP30 as a function of N/P ratios against SKOV-3 cells in the absence of serum; a polyplex formulation of BpEI and L-pEI at an optimal N/P ratio of 8/1 or Lipo 2000 at an optimal formulation of 2 μL/μg DNA was used as a control; (d) transfection efficiency of Dex-SSBAP6 and Dex-SSBAP30 at an N/P ratio of 10/1 against SKOV-3 cells in the absence (w/o) or presence (w/) of 10% FBS. (*P < 0.05; NS, not significant).

that these disulfide-based cationic dextran conjugates may facilitate intracellular gene release, as similar to pSSBAP (Figure 1b). To ascertain whether Dex-SSBAP6 and Dex-SSBAP30 are applicable for gene delivery, their in vitro transfection activity and cytotoxicity were investigated. It was showed that these two conjugates had a low cytotoxicity against SKOV-3 and MCF-7 cells, with more than 90% cell viability at a high polymer concentration of 100 mg/L (Figure 3a,b). This low cytotoxicity could benefit from low-toxic dextran and biodegradability of disulfide-linked BAP side chain. As a negative control, B-pEI revealed high cytotoxicity with IC50 of ∼25 mg/L. Next, in vitro transfection activity of the conjugates was evaluated against SKOV-3 cells with the plasmid DNA encoding luciferase reporter gene. Transfection efficiency of the conjugates was optimized as a function of N/P ratios after the cells were transfected by their polyplexes in the absence of serum. At the same N/P ratio of 10/1, the polyplexes of Dex-SSBAP30 yielded higher transfection efficiency as compared to those of Dex-SSBAP6 (Figure 3c). Moreover, the polyplexes of DexSSBAP30 at an N/P ratio of 30/1 afforded comparable transfection efficiency to those of L-pEI and lipofectamine 2000 (Lipo 2000) but lower transfection efficiency than those of BpEI. Further, the effect of serum on the transfection efficiency of Dex-SSBAP6 and Dex-SSBAP30 was examined at an N/P ratio of 10/1. Interestingly, 10% FBS did not significantly influence the transfection efficiencies of the conjugates (Figure 3d). However, the FBS markedly decreased the efficiencies of B-pEI and L-pEI by ca. 4.7 and 5.3 times, respectively, compared to those obtained without FBS. Taken together, DexSSBAP30 is an ideal candidate for systemic gene delivery due to good colloidal stability of its polyplexes (Figure 2b) and efficient gene transfection in serum. 3.3. Dex-SSBAP Conjugate and Folate-Coupled DexSSBAP for i.v. Gene Delivery. Folate (FA), a high-affinity

SSBAP conjugates with different amounts of BAP residues can be prepared successfully. Particle size and surface charge of the polyplexes of DexSSBAP6 and Dex-SSBAP30 were characterized by DLS analysis (Figure 2a,b). The conjugates can efficiently bind DNA to form nanoscale polyplexes with positive surface charges at the N/P ratios from 10/1 to 30/1. TEM imaging again confirmed the formation of nanoscale polyplexes through complexation of Dex-SSBAP30 and DNA (Figure S7, Supporting Information). Notably, at the same N/P ratio of 10/1, the polyplexes of DexSSBAP6 and Dex-SSBAP30 displayed a lower surface charge (+11 and +16 mV) as compared to those of pSSBAP (+23 mV), which is likely attributed to the presence of neutral dextran segment surrounding the polyplexes. It is expected that the dextran in the conjugates may endow their polyplexes with an improved colloidal stability in physiological conditions. To proof this, colloidal stability of the polyplexes in HEPES buffer saline (20 mM and 150 mM NaCl) was assessed by measuring particle size as a function of time ranging from 0.5 to 4 h (Figure 2c). It was found that the polyplexes of Dex-SSBAP6 displayed an improved colloidal stability at 0.5 h, but formed large particles of ∼1000 nm at 4 h (Figure S8a, Supporting Information). The polyplexes of Dex-SSBAP30, however, had better colloidal stability because their particle sizes kept stable (less than 250 nm) within 4 h (Figure S8b, Supporting Information). By contrast, the size of the polyplexes of pSSBAP rapidly increased to ∼500 nm at 0.5 h and ∼1200 nm at 4 h. Thus, an appropriate composition ratio of BAP residues and dextran in Dex-SSBAP conjugates is relevant to afford polyplexes with an improved colloidal stability. DNA binding and release profiles of the cationic dextran conjugates were also examined by agarose gel retardation analysis (Figure 2d). DexSSBAP6 and Dex-SSBAP30 caused a complete retardation of DNA mobility at and above an N/P ratio of 1/1. However, the presence of DTT led to an efficient DNA release, suggesting H

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ligand for folate receptor,33 was coupled to primary amine of Dex-SSBAP30 by carbodiimide chemistry, yielding FA-coupled Dex-SSBAP30 (denoted as Dex-SSBAP30-FA, Scheme 3). 1H NMR spectra of Dex-SSBAP30-FA exhibited three new signals at δ 6.8, 7.7, and 8.8 (Figure 4), attributed to the protons of FA,

injection of the polyplexes of Dex-SSBAP30 or Dex-SSBAP30FA conjugates at an optimal N/P ratio of 10/1 and using the plasmid DNA encoding luciferase gene. It was found that the polyplexes of Dex-SSBAP30-FA afforded about 2−3 times higher gene expression in tumor than other normal organs (Figure 5d). Moreover, the polyplexes afforded ca. 4.3 times higher gene expression in the tumor as compared to those of Dex-SSBAP30 without the folate. This higher efficacy is probably due to efficient uptake of the polyplexes into SKOV-3 cells by FA receptor-mediated endocytosis. Although Dex-SSBAP30 lacks FA, its polyplexes also produced about 2− 7 times higher gene expression in the tumor than other organs, which is likely attributed to passive targeting of the polyplexes in the tumor by enhanced permeation and retention (EPR) effect.34 Both Dex-SSBAP30-FA and Dex-SSBAP30 induced detectable gene expression in spleen and liver, although lower than that in the tumor, indicating that some of their polyplexes finally suffer from clearance by the organs. In order to provide the evidence on in vivo targeting activity of Dex-SSBAP30-FA, we investigated accumulation kinetics of Cy5.5-labeled polyplexes in SKOV-3-tumor bearing in nude mice. Three-dimensional-in vivo near-infrared fluorescence imaging system clearly exhibited stronger fluorescence signal for Dex-SSBAP30-FA group compared to Dex-SSBAP30 group at 2 h postinjection (Figure 5e). Also, quantification analysis of the signal intensity confirmed that the amount of DexSSBAP30-FA was ca. 3 times higher than that of DexSSBAP30. Besides, ex vivo fluorescence imaging of isolated tumors showed that, even at 24 h postinjection, Dex-SSBAP30FA retained accumulation at a larger level when compared to Dex-SSBAP30 (Figure 5f). These results imply efficient in vivo targeting of Dex-SSBAP30-FA to SKOV-3 tumor. Notably, at 24 h postinjection, stronger signal intensity (Figure 5f) was also detected in the organs, i.e., spleen and liver for Dex-SSBAP30-FA group than for Dex-SSBAP30 group, reflecting an enhanced elimination of Dex-SSBAP30-FA-based polyplexes by the organs. As revealed in Figure S10, Supporting Information, ex vivo fluorescence imaging analysis showed that, at 2 h postinjection, Dex-SSBAP30-FA displayed a higher level of uptake in liver and spleen as compared to Dex-SSBAP30. This higher uptake might account for the finding that DexSSBAP30-FA induced a higher level of transgene expression in liver and spleen than Dex-SSBAP30. A similar event was also found by Gabizon et al.35,36 and McNeeley et al.,37 who found about 2-fold higher uptake in liver for folate-liposomes compared to nontargeted counterparts. A possible interpretation on the enhanced clearance of folate-modified particles (liposomes/polyplexes) by the liver could be due to their active interactions with folate receptors in the liver, or their binding with the plasma folate binding proteins, which facilitates opsonization and particle removal by a mononuclear phagocyte system. L-pEI (22 kDa) was used as a positive control (Figure 5d) since it was more efficient for in vivo gene delivery than B-pEI.38 Compared to L-pEI, Dex-SSBAP30-FA led to comparable transfection efficacy in the tumor, but ca. 2-folded lower in the lung. It was shown that L-pEI induced gene expression not only in the tumor due to EPR effect,34 but in the lung because its unstable polyplexes tend to form large-sized aggregates, which are mainly accumulated in the abundant capillary network of lung.39 However, the polyplexes of Dex-SSBAP30 conjugate displayed an improved colloidal stability under physiological conditions (Figure 2c), which may favorably contribute to

Figure 4. 1H NMR spectra of folate-modified disulfide-based cationic dextran (Dex-SSBAP30-FA).

implying successful conjugation of FA to Dex-SSBAP30. Besides, from the spectra, the amount of FA residue in this conjugate was determined to be ∼3 per dextran chain, by comparing the integrals of the peak at δ 6.8 (aromatic proton in FA) and δ 4.95 (anomeric proton in dextran). In vitro transfection activity of Dex-SSBAP30 and DexSSBAP30-FA was investigated against SKOV-3 cells, overexpressing FA receptor, in the presence of serum, and transfection efficiency was optimized as a function of N/P ratios from 10/1 to 30/1 (Figure 5a). It was found that the transfection efficiency of Dex-SSBAP30-FA was not dependent on the N/P ratios and that the optimal efficiency was obtained at the N/P ratio of 10/1. However, the transfection efficiency of Dex-SSBAP30 was augmented by ca. 2 times with increasing N/P ratios from 10/1 to 30/1. Moreover, at the same N/P ratio of 10/1, the polyplexes of Dex-SSBAP30-FA afforded ca. 2-folded higher efficiency as compared to those of DexSSBAP30. These results reflect that the polyplexes of DexSSBAP30 are uptaken into SKOV-3 cells by adsorptive endocytosis, and the polyplexes of Dex-SSBAP30-FA are, however, uptaken by FA receptor-mediated endocytosis. Support for this hypothesis is also found by cellular uptake assay of the polyplexes of Dex-SSBAP30-FA in free FA. As shown in Figure 5b, fluorescence histogram of SKOV-3 cells, treated by FA (200 μM), clearly shifted to a weaker histogram region as compared to that of untreated cells (control). This suggests that free FA inhibits uptake of the polyplexes, i.e., ca. 13% of cellular uptake in comparison with that of untreated cells (Figure 5c). By contrast, free FA did not affect uptake of the polyplexes of Dex-SSBAP30 as a negative control. This pronounced inhibitory effect on uptake of the polyplexes of Dex-SSBAP30-FA may explain why free FA leads to reduced transfection efficiency of the polyplexes (Figure S9, Supporting Information). Systemic gene delivery of Dex-SSBAP conjugates was investigated in Balb/c nude mice bearing SKOV-3 tumor. Biodistribution of gene expression in normal organs and tumor was determined by luciferase expression assay 1 day after i.v. I

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Figure 5. (a) Transfection efficiency of Dex-SSBAP30-FA against SKOV-3 cells in the presence of 10% FBS; (b) fluorescence histogram of SKOV-3 cells before and after treated by free folate (200 μM); (c) effect of free folate (100−400 μM) on uptake of Dex-SSBAP30-FA/YOYO-1 labeled DNA polyplexes at an N/P ratio of 10/1; (d) Tumor and organ luciferase gene expression, 24 h after i.v. injection of polyplexes of Dex-SSBAP30 or DexSSBAP30-FA containing pCMV-Luc (50 μg) at an N/P ratio of 10/1 in a SKOV-3 tumor xenografted Balb/c nude mouse model (n = 5). The polyplexes of L-pEI containing pCMV-Luc (50 μg) at an optimal N/P ratio of 10/1 were used as a positive control. (*P < 0.05); (e) 3D-in vivo nearinfrared fluorescence imaging of Cy5.5-labeled polyplexes based on Dex-SSBAP30-FA or Dex-SSBAP30, formed at an N/P ratio of 10/1, in SKOV-3 tumor bearing nude mice at 2 h postinjection; tumor accumulation (%ID) of the polyplexes as a function of postinjection time was also shown after quantification of fluorescence signal intensity; (f) 2D-ex vivo imaging of Cy5.5-labeled polyplexes in tumor and organs at 24 h postinjection.

those of L-pEI (Figure 7). Although both two groups showed almost the same 60% of ID% in the plasma at 2 min postinjection, at 80 min postinjection 8.95% of Dex-SSBAP30FA retained in the plasma, while L-pEI was largely eliminated from the circulation (2.21%). This result suggests that this cationic dextran conjugate is more suitable for i.v. administration than L-pEI. One feasible interpretation on the prolonged circulation is, at an N/P ratio of 10/1, the polyplexes of Dex-SSBAP30-FA have lower positive surface charge when compared to those of L-pEI (+15.5 ± 0.3 vs. +20.4 ± 0.5 mV), which minimizes nonspecific interactions with the blood cells. In a previous report, Kissel et al. showed that the polyplexes of branched PEI displayed fast blood clearance profile (10−20% of ID% in the plasma at 2 min post-i.v. injection) as a result of their high positive surface charge (+26 ± 2 mV).41 Further studies will be focused on optimizing polycation/dextran composition of this cationic dextran conjugate system to modulate surface charge as well as evaluate biodistribution, circulation, and gene expression in mice. Overall, folate-

diminished accumulation in the lung and subsequently a low gene expression. Systemic gene delivery of Dex-SSBAP30-FA was also evaluated by i.v. injection of its polyplexes with the plasmid DNA encoding LacZ gene in the same nude mouse model. Beta-galactosidase expressed in tissues was visualized by X-Gal staining. As revealed in Figure 6, Dex-SSBAP30-FA indeed induced gene expression (pale blue) in the tumor and other normal organs (except heart). For example, the gene expression in the lung was clearly observed in the bronchial epithelial cells (Figure 6d). This phenomenon was also found in those previous reports,39,40 where L-pEI or dextran− spermine conjugates led to gene expression in the epithelial cells. PEGylated polyplexes are found to be suited for systemic gene delivery as a result of their prolonged circulation property. However, it is not elucidated if dextranated polyplexes are effective for a long-term circulation. Circulation kinetics of polyplexes of Cy5.5-labeled Dex-SSBAP30-FA confirmed that the polyplexes had improved circulation profile compared to J

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Figure 6. X-gal staining of LacZ expression in tumor and organs, 24 h after i.v. injection of the polyplexes of Dex-SSBAP30-FA containing pCMVLacZ (50 μg) at an N/P ratio of 10/1 in a SKOV-3 tumor-bearing Balb/c nude mouse model.

SKOV-3 tumor bearing in a nude mouse model by i.v. injection, causing a high level of gene expression in the tumor. Thus, disulfide-based cationic dextran conjugates may be finely designed as multifunctional gene delivery vectors to afford the polyplexes with an improved colloidal stability, redoxtriggered gene release and targeting ability, and has a great promise for use in cancer gene therapy.

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ASSOCIATED CONTENT

* Supporting Information S

1

H NMR spectrum, acid−base titration curve, and TEM imaging of polymers and cationic dextran conjugates. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Figure 7. Blood level curves of the polyplexes of Dex-SSPBA30-FA (or L-pEI) as a control, prepared at an N/P ratio of 10/1 and 30 μg of DNA. ***P < 0.001: Dex-SSPBA30-FA vs. L-pEI (n = 4−6 mice).

AUTHOR INFORMATION

Corresponding Author

*(C.L.) E-mail: [email protected]. Tel: 0086-2165988029. Fax: 0086-21-65983706-0.

modified disulfide-based cationic dextran conjugate may serve as a multifunctional gene vector for systemic (i.v.) gene delivery targeting ovarian cancer cells.

Author Contributions §

(Y.S. and B.L.) These authors contributed equally to this work.

Notes

4. CONCLUSIONS We have showed that bioreducible cationic dextran conjugates can be prepared that have linear dextran as the main chain and disulfide-linked 1,4-bis(3-aminopropyl)piperazine (BAP) residues as the side chains. These cationic dextran conjugates having about 6 and 30 BAP residues (Dex-SSBAP6 and DexSSBAP30, respectively) are able to condense DNA to form nanoscale polyplexes with an improved colloidal stability and moderate surface charge in physiological conditions. The polyplexes are, however, redox-sensitive to liberate DNA in an intracellular reducing environment. Dex-SSBAP30 conjugate is capable of gene transfection in vitro, inducing comparable efficiency to that of L-pEI in SKOV-3 cells in 10% serum. Importantly, the conjugates have low cytotoxicity in vitro against SKOV-3 and MCF-7 cells. A unique merit is that the conjugates have a primary amine terminal group at their side chain and thus allow for further chemical decoration with a homing device such as folate. As such, folate-coupled DexSSBAP30 conjugate can be generated and mediate highly efficient gene delivery targeting SKOV-3 cells in vitro and

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Shanghai Municipal Natural Science Foundation (13ZR1443600), National Basic Research Program of China (973 program) (2014CB964600 and 2012CB966300), National Natural Science Foundation of China (20904041), National High Technology Research and Development Program 863 (SS2013AA031902), and Fundamental Research Funds for the Central Universities (to C.L.).

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REFERENCES

(1) Anderson, W. F. Human Gene Therapy. Nature 1998, 392, 25− 30. (2) Thomas, C. E.; Ehrhardt, A.; Kay, M. A. Progress and Problems with the Use of Viral Vectors for Gene Therapy. Nat. Rev. Genet. 2003, 4, 346−358. (3) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Design and Development of Polymers for Gene Delivery. Nat. Rev. Drug Discovery 2005, 4, 589−593. K

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dx.doi.org/10.1021/mp4006672 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX