iMDR1-pDNA complex nanoparticles

Biomaterials 32 (2011) 1738e1747

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Reversal of multidrug resistance by reduction-sensitive linear cationic click polymer/iMDR1-pDNA complex nanoparticles Yu Gao, Lingli Chen, Zhiwen Zhang, Yi Chen, Yaping Li* Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 August 2010 Accepted 1 November 2010 Available online 26 November 2010

A reduction-sensitive linear cationic click polymer (RCP) was specially designed for the efficient gene delivery to overcome multidrug resistance (MDR) by RNA interference to silence the expression of Pglycoprotein (P-gp). RCP was synthesized via the “click chemistry” with disulfide bonds, amideetriazole moieties and secondary amine groups in the main chain. RCP could efficiently condense pDNA into nanoparticles (RCPNs) around 150 nm. Polyplex dissociation was observed in the presence of 2.5 mM DTT due to the cleavage of disulfide bonds, which indicated the efficient DNA release under the reduction condition. In vitro transfection and cytotoxicity experiments against human breast cancer MCF-7 cells and drug-resistant MCF-7/ADR cells showed that RCPNs could bring about higher transfection efficiency with much lower cytotoxicity than PEI/DNA nanoparticles. RCPNs loaded with plasmid iMDR1-pDNA could inhibit P-gp expression, increase adriamycin (ADR) accumulation and enhance cytotoxicity of ADR against MCF-7/ADR cells. Combination of RCPNs and ADR could suppress the tumor growth more efficiently than using ADR only on mouse xenograft model bearing ADR resistant human breast cancer. These results suggested that this RCP could be a potential, safe and efficient non-viral vector for reversing MDR. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Click chemistry RNA interference Disulfide bond P-glycoprotein Multidrug resistance

1. Introduction Breast cancer is the most common cancer in women and the second leading cause of cancer death after lung cancer [1]. Although the chemotherapy is an important approach to treat breast cancer, the development of multidrug resistance (MDR) becomes a serious obstacle to the effective chemotherapy [2,3]. One of the best known mechanisms responsible for the MDR phenotype in breast cancer is the overexpression of P-glycoprotein (P-gp) encoded by MDR1 gene in cancer cells [4e6]. Generally, P-gp acts as export “pumps” for a wide range of structurally and functionally unrelated chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins and taxanes. Therefore, the inhibition of P-gp mediated drug efflux could be one of the most effective ways to re-sensitize tumor cells to chemotherapy to reverse MDR. Up to now, several MDR reversing agents, such as verapamil, quinidine and VX-710, have been developed to inhibit the function of P-gp [7]. Unfortunately, the related issues such as the high inherent toxicity of P-gp inhibitors and the changed pharmacokinetic of anticancer drugs co-administered with P-gp inhibitors limited their application in clinical trials [8,9]. Rather than blocking the drug pump function of P-gp, the inhibition of P-gp expression could be a more * Corresponding author. Tel./fax: þ86 21 20231979. E-mail address: [email protected] (Y. Li). 0142-9612/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.11.001

efficient and specific strategy to conquer MDR. In recent years, RNA interference (RNAi) has been widely used as a new approach to silence gene expression by an efficient and specific way [10]. Because pDNA or siRNA could not penetrate the cellular membrane efficiently and be easily degraded in vivo, a safe and efficient gene delivery vector is needed to protect them from nuclease degradation and efficiently deliver them into tumor cells to exert their silencing effects. Compared with viral vectors, non-viral vectors present certain advantages with simple large scale production, low host immunogenicity, the possibility selected modifications and the capacity to carry large inserts [11,12]. Cationic polymers which are in the context of non-viral gene delivery have drawn the increasing attention in recent years. However, the contradictions between transfection efficiency and cytotoxicity of polymers, and the DNA condensation efficiency of polymer outside cells and the easy dissociation of DNA from polymer in nucleus are two of the difficult problems in polymer-based gene delivery systems [13e15]. Disulfide linkage contained in the polymer backbone which can be rapidly cleaved in the presence of high concentrations of glutathione (GSH) could be one of the best solutions to the above-mentioned problems. The intracellular glutathione concentration is 50e1000 times higher than the extracellular concentration [16]. Complex nanoparticles with disulfide could keep stable in extracellular environment, while in intracellular environment, the entrapped pDNA could efficiently release from the complex nanoparticles with disulfide degradation. In addition, the presence of

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disulfide linkage in the polymer backbone could increase polymer biodegradability and reduce polymer toxicity. Recently, the copper-catalyzed azideealkyne ligation process has been successfully employed in the linear polymer synthesis [17]. The cationic polymers synthesized by click chemistry have demonstrated great potential for gene delivery [18,19]. The amideetriazole moiety, which was formed during polymerization, could act as H-bond acceptors and increase hydrophobic interactions of the polymer with DNA, thus improve the polymereDNA binding stability [18,20,21]. We are interested in developing a safe and efficient gene vector for delivery of pDNA expressing shRNA targeting MDR-1 gene (iMDR1-pDNA) into MDR tumor cells to reverse MDR. In this work, we firstly introduced disulfide bond into linear click polymer (CP) to synthesize reduction-sensitive linear cationic CP (RCP) that utilized the advantages of both amideetriazole moiety and disulfide bond. The in vitro and in vivo reversal of P-gp mediated MDR by RCP/iMDR1-pDNA complex nanoparticles (RCPNs) were investigated in adriamycin (ADR) resistant MCF-7/ADR cells and MDR human breast cancer xenografts in nude mice.

dithiodipropionic acid, 1-chloro-3-aminopropane hydrochloride, ethyl trifluoroacetate and branched polyethylenimine (PEI, 25 kDa) were purchased from Sigma (St. Louis, MO, USA). Phycoerythrin (PE)-anti-human MDR1 (CD243, P-gp, ABCB1) and PE-Mouse IgG2a (K Isotype Control) were obtained from eBioscience (CA, USA). Rabbit anti-human P-gp and HRP conjugated goat anti-rabbit secondary antibody were purchased from Santa Cruz (CA, USA). Adriamycin (ADR) was purchased from Hisun Pharmaceutical Co. Ltd. (Zhejiang, China). All other chemicals and solvents if not mentioned were of analytical grade and used as received without additional purification. Plasmid EGFP-N1（4.7 kb） encoding enhanced green fluorescent protein driven by immediate early promoter of CMV was purchased from Clontech (Palo Alto, CA, USA). The anti-MDR1 shRNA expression system (iMDR1-pDNA) was constructed as described previously [22]. Scrambled DNA used as negative control (neg-DNA) has random sequences with no homology. The plasmid DNA (pDNA) grown in DH5a strain of E. coli was isolated with the EndFree Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). The purity was confirmed by spectrophotometry (A260/A280), and DNA concentration was measured by UV absorption at 260 nm.

2. Materials and methods

2.3. Animals

2.1. Materials

Female nude mice (18e20 g, 6 weeks old) were purchased from Shanghai experimental animal center (Shanghai). All animal procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Triethylenetetramine, diethylene glycol, 4-dimethylamino pyridine (DMAP), copper (II) sulfate, trifluoroacetic acid and 4-toluene sulfonyl chloride were got from Sinopharm Group Chemical Reagent Co., Ltd. (China). N,N0 -dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC$HCl) and N-Hydroxysuccinimide (NHS) were purchased from GL Biochem Ltd. (Shanghai, China). Trypsin-EDTA, phosphate buffered solution (PBS) and agarose were obtained from Gibco-BRL (Burlington, ON, Canada). The Dulbecco’s modified Eagle medium (DMEM), antibiotics, DNA loading buffer and fetal bovine serum (FBS) were purchased from Invitrogen GmbH (Karlsruhe, Germany). Ethidium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dithilthreitol (DTT), 3,30 -

O

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2.2. Cell culture The cell lines MCF-7 (human breast cancer cell) and ADR resistant MCF-7/ADR cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in DMEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin G sodium and 100 mg/ml streptomycin sulfate and maintained at 37  C in a humidified and 5% CO2 incubator.

2.4. Synthesis of monomers The structures of monomers used in the synthesis of RCP were shown in Fig. 1. Monomer (1-azido-2-(2-azidoethoxy)ethane) (2) was synthesized as follows, to a stirred solution of diethylene glycol (2.12 g, 20 mmol) in dichloromethane (250 mL) at 0  C, triethylamine (4.25 g, 42 mmol), DMAP (0.12 g, 0.1 mmol) and 4-toluene sulfonyl chloride (7.62 g, 42 mmol) were added. The mixture was stirred at 0e10  C

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Fig. 1. Scheme for synthesis of RCP.

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for 1 h, then at room temperature for 12 h. The reaction solution was extracted with saturated NaHCO3 solution (2  300 mL) and then with saturated NaCl solution (300 mL). The organic layers were dried over Na2SO4, and the dichloromethane was evaporated to yield the off-white solid. The crude product was purified by recrystallization from CH2Cl2/petroleum ether to yield white solid 2,20 -oxybis(ethane-2,1diyl) bis(4-methylbenzenesulfonate). To a solution of 2,20 -oxybis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate) (4.1 g, 10 mmol) in acetone, sodium azide (1.9 g, 30 mmol) was added, and the mixture was refluxed for 48 h. After evaporating the solvent, the resulting crude product was purified via silica gel chromatography using an elution of 50% ethyl acetate in petroleum ether (Rf ¼ 0.8 in 50% ethyl acetate/ petroleum ether). The proper fractions were combined, and the solvent was evaporated to yield 2 as yellow oil. 1H NMR (CDCl3): 2.87 (s, 4H), 2.70 (s, 4H). Monomer tert-butyl bis (2-propiolamidothyl) carbamate (4) was obtained using a four-step synthesis procedure as reported previously [18]. The di-azide monomer containing a disulfide bond (6) was synthesized by conjugation 3,30 -dithiodipropionic acid (5) with 1-azido-3-aminopropane. Firstly, 1-azido-3-aminopropane was synthesized as previously reported [23]. Secondly, 1-azido-3-aminopropane was reacted with 5 to form monomer 6. Briefly, to a stirred solution of 5 (2.1 g, 10 mmol) in water (100 mL), EDC$HCl (4.34 g, 22 mmol) and NHS (2.53 g, 22 mmol) were added and stirred for 2 h. To this mixture, a solution of 1-azido-3-aminopropane (2.2 g, 22 mmol) dissolved in 25 mL water was added, and the reaction mixture was then stirred for 24 h. The reaction mixture was extracted with ethyl acetate. The organic layers were combined and dried over Na2SO4. The ethyl acetate was evaporated, and the resulting crude product was purified via silica gel chromatography using a gradient elution of 2%e4% methanol in chloroform (Rf ¼ 0.25 for 6 in 4% MeOH/CHCl3). The proper fractions were combined, the solvent evaporated, and the resulting white solid was isolated. 1H NMR (CD3OD): 8.19 (bs, 2H), 3.36 (t, J ¼ 5.2 Hz, 4H), 3.25 (t, J ¼ 6.0 Hz, 4H), 2.94 (t, J ¼ 7.2 Hz, 4H), 2.59 (t, J ¼ 6.8 Hz, 4H), 1.78e1.72 (m, 4H). 2.5. Synthesis and characterization of RCP The synthesis procedure of RCP was shown in Fig. 1. The solution of copper (II) sulfate (0.2 eq) in water was added to the solution of monomer 4 (1 eq), monomer 2 (0.9 eq), and monomer 6 (0.1 eq) dissolved in tert-butanol, and the mixture was introduced into a small vial covered with a rubber cap. The vial was filled with nitrogen in vacuum. The solution of sodium ascorbate (0.4 eq) dissolved in water was added dropwise with a thin pinhead piercing through the rubber cap. The mixture was heated to 50  C in an oil bath, stirred for 24 h, and then cooled with an ice bath. The solvents were evaporated and the resulting copolymer was then dissolved in 10 mL of a solution of dichloromethane/trifluoroacetic acid (1:1 volume), stirred for 3 h to remove the Boc protecting groups, and dried under vacuum. The final product after neutralized using 1 M NaOH solution was purified via dialysis against 0.01 M HCl for 24 h, and then exhaustively against ultra pure H2O for 3 days. The final deprotected polymers were lyophilized to dryness to yield a brown solid. The 1H spectra were recorded on a Bruker 400 MHz NMR Spectrometer. Gel permeation chromatography (GPC) was conducted on a Waters 2695 controller equipped with ULTRAHYDROGEL columns (250 PKGD and 1000 PKGD) and a refractive index detector (model 2414) to estimate the polymerization degree and polydispersity (Mw/Mn) of click polymer. The polymer was dissolved in water (0.05% sodium azide), and the sample was analyzed and calibrated by DEXTRAN standards (Mp ¼ 4400e124000 Da, American Polymer Standards Corporation, USA). 2.6. Preparation and characteristics of RCP/DNA nanoparticles (RCPNs) RCPNs were prepared by the following procedures. Briefly, pDNA (plasmid EGFPN1, 100 mg/ml) was added to RCP solution diluted in sterile water with equal volume to obtain the desired polymer-amine to DNA-phosphate ratio (N/P ratio). The polymer-amine was calculated from the amine groups in the polymer backbone, and the amide and triazole nitrogens were not considered in the N/P ratio calculation. 330 Da was used as an average mass per charge for DNA. Subsequently, solution was immediately vortexed for 30 s (XW-80A Votex mixer, Shanghai). The resulting complexes were allowed to sit at room temperature for 30 min. RCPNs complexed with iMDR1-pDNA used to investigate reversal of MDR in tumor cells were prepared with the same procedure. As positive control, PEI25K/DNA complex nanoparticles (PEINs) were prepared with the same procedure as RCPNs. The particle size and zeta potential of RCPNs were determined by the laser light scattering measurement using a Nicomp 380/ZLS zeta potential analyzer (Particle Sizing System, USA). The RCPNs were prepared as described above, and the size measurement was performed at 25  C at a 90 scattering angle, recorded for 300 s for each measurement. To determine the reduction-sensitivity of RCPNs, particle sizes of complex nanoparticles after treated with DTT (2.5 mM) for 10 min were also measured. The AFM images were obtained by depositing 10 ml of RCPNs samples onto the center of a freshly cleaved mica disk and allowing the solution to dry in air for 10 min at room temperature and image immediately. AFM imaging was performed in air with a Multimode Pico-Force AFM (Veeco Instruments Inc., Santa Barbara, CA) using silicon tapping tips (NP-S20, Veeco Instruments). Complex formation and reduction-sensitivity of RCPNs were also confirmed by agarose gel electrophoresis. RCPNs were first prepared as described above. Next, 20 ml of 25 mM DTT solution or sterile water (as control) was added to 180 ml of

RCPNs, and the dispersions were incubated for 30 min. After addition of DNA loading buffer, RCPNs containing 0.1 mg DNA at various N/P ratios were applied to a 1.0% agarose gel in Tris-Acetate-EDTA (TAE) buffer containing 0.5 mg/ml ethidium bromide. Electrophoresis was carried out at 80 V for 45 min in TAE buffer. The resulting DNA migration pattern was revealed under UV irradiation. 2.7. In vitro transfection and cytotoxicity of RCPNs Cells (MCF-7 and MCF-7/ADR) were seeded in 24-well plates at a density of 1  105 cells per well in 500 ml of complete medium and incubated for 24 h prior to transfection. Then, the media were replaced with fresh complete growth medium containing RCPNs with DNA concentration 2.5 mg/well at various N/P ratios. Media were replaced by fresh culture medium after 24 h, and cells were incubated for an additional 24 h. The transfection results were measured using a FACSCalibur through FL1. As control, naked DNA was used on cell cultures and examined as described above. As positive control, PEINs with mass ratio 3 were prepared and transfected with media without FBS for 2 h before the medium was replaced with fresh complete growth medium. The cytotoxicity was evaluated by MTT assay. MCF-7 and MCF-7/ADR cells were seeded at a density of 1  104 cells per well in 150 ml growth medium in 96-well plates and grown overnight. Immediately after growth medium was removed, RCPNs were applied onto the cells in fresh culture media with 10% FBS at polymer concentrations ranging from 5 to 200 mg/ml. Cells treated with fresh culture media only were used as control. Incubations were made for 2 and 24 h before removal of media containing polymers and replacement with fresh culture media. After 24 h, the media were replaced with fresh culture media containing MTT solution (0.5 mg/ ml), and the cells were incubated for an additional 4 h at 37  C. Then, the medium was removed, and DMSO was added to dissolve the crystal. The plates were mildly shaken for 10 min to ensure the dissolution of formazan. The absorbency values were measured by TECAN infinitÔ F200 multimode microplate reader (Salzburg, Austria) at wavelength 570 nm, blanked with DMSO solution. Six replicates were counted for each sample. 2.8. Determination of P-gp MCF-7 and MCF-7/ADR cells were seeded into 24-well plates at a density of 1  105/well with 0.5 ml growth medium and allowed to attach for 24 h. The media was replaced with fresh growth medium containing RCPNs with DNA concentration 2.5 mg/well at N/P ratio 32. After 24 h incubation, media were replaced by fresh culture medium, and cells were incubated for an additional 24 h. Cells were trypsinized, collected and resuspended in PBS (pH 7.4). PE-conjugated mouse anti-human monoclonal antibody against P-gp was used to label cells according to manufacturer’s instruction, and the nonspecific labeling was corrected by its isotype control. The cell resuspension was finally subjected to a FACS Caliber (Beckton Dickinson, USA) and analyzed with CellQuest software through fluorescence channel 2 (FL2). For western blot analysis, MCF-7 and MCF-7/ADR cells were seeded into 6-well plates at a density of 5  105/well with 2 ml growth medium and allowed to attach for 24 h. After transfection, cells were treated with lysis buffer and the total protein concentration was determined by a UVeVis spectrophotometer. The samples were then mixed with 5SDS sample loading buffer (Invitrogen) and boiled for 10 min. Samples of 50 mg of total protein per lane were separated on 8% SDS-PAGE. The resultant proteins were blotted onto NC membrane. The membrane was incubated with skimmed milk at room temperature for 3 h, then incubated with rabbit anti Pgp antibody diluted 1:200 (v/v) and rabbit anti b-actin antibody at room temperature for 3 h and at 4  C overnight. After incubated with HRP conjugated goat antirabbit secondary antibody for 1.5 h at room temperature, the membrane was exposed to Kodark film using ECL Plus reagent, and the protein bands were determined. b-actin was served as an internal standard protein. 2.9. ADR uptake assay MCF-7/ADR cells were seeded into 24-well plates at a density of 1 105/well with 0.5 ml growth medium and allowed to attach for 24 h. Cells treated or not treated with RCPNs were incubated with 1 mg/ml ADR for 2 h. After replacement with flesh culture medium, cells were allowed to efflux ADR for 2 h. Then, cells were collected, resuspended in PBS (pH 7.4) and immediately analyzed with flow cytometry through FL2. For confocal observation, MCF-7/ADR cells were seeded on 35-mm glass-based dishes with 0.5 ml growth medium and allowed to attach for 24 h. Cells treated or not treated with RCPNs were incubated with 1 mg/ml ADR for 2 h. After replacement with flesh culture medium, cells were allowed to efflux ADR for 2 h. Then, the cells were washed three times with PBS and observed by confocal laser scanning microscopy (CLSM). The CLSM observation was performed using FLuoViewÔ FV1000 confocal microscope (Olympus Microsystems, Japan) with a 40 objective. 2.10. Antiproliferation activity of ADR against MCF-7/ADR cells MCF-7/ADR cells were seeded at a density of 1  104 cells per well in 150 ml growth medium in 96-well plates and grown overnight. Immediately after growth medium was removed, RCPNs (N/P ratio 32) were applied onto the cells in fresh

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culture media at DNA concentration 2.5 mg/well. Cells treated with fresh culture media only were used as control. After 24 h incubation, the complex-containing media were replaced with fresh culture media containing different concentration of ADR. After 48 h incubation, cell media were replaced with fresh medium and subjected to MTT analysis. 2.11. In vivo therapeutic experiment Balb/c nude mice were inoculated with 100 ml of phosphate buffered saline (pH 7.4) containing 1  107 MCF-7/ADR cells by subcutaneous injection in the left flank. Experiments were performed at day 10 after inoculation when the tumor volume reached approximately 100 mm3. Tumor-bearing mice were randomly assigned to the following four treatment groups (n ¼ 6): saline control group, RCPNs group (2 mg DNA/ kg), ADR alone group (8 mg/kg) or combination of RCPNs and ADR group. Mice were administered through tail vein once a week for 3 weeks, and ADR was given at 48 h after administration of RCPNs. Animals’ body weight and the tumor volume ([major axis]  [minor axis]2  1/2) were measured with calipers twice a week over a period of 21 days. Tumor volume on the day of treatment was normalized to 100% for all groups. 2.12. Statistical analysis Statistical analysis was performed using a Student’s t-test. The differences were considered significant for p value <0.05 and p < 0.01 indicative of a very significant difference.

3. Results 3.1. Synthesis and characterization of RCP In this work, two azide-functionalized monomers (2 and 6) and one alkyne-functionalized monomer (4) were used to synthesize RCP. Monomer 6 contained a disulfide bone that was functionalized with azide was used to introduce disulfide bond into RCP using “click chemistry”. The 1H NMR spectrum of monomer 6 was shown in Fig. 2A. Monomer 4 was designed with second amine to endow polymer with cationic property. Monomer 2 was used to reduce charge density and make polymer with more hydrophilicity. In previous reports, the syntheses of linear click polymer were all based on one kind or two kinds of monomers at equivalent molar ratio [17,18,24]. We originally intended to use monomer 4 and monomer 6 to synthesize click polymer containing disulfide bond, however, the water solubility of the obtained polymer was very poor. Therefore, monomer 2 was added to the chemical reaction to synthesize RCP by “one-pot synthesis” strategy. We conducted the experiments under the condition of a series of monomer ratios of monomer 2 to 6, and found that the solubility of click polymer decreased with the ratio of monomer 6 increasing. Considering that it could increase polymer solubility by increasing the content of monomer 2, and it also could make polymer with reductionsensitivity by introducing only a certain amount of monomer 6, the molar ratio of monomer 2 to 6 was set at 9:1. The RCP synthesized from monomer 2, 4 and 6 at molar ratios of 9:10:1 was used for the rest of the experiments. In polymer synthesis, solution of tert-butyl alcohol and water (1:1, v/v) was chosen as a general solvent for polymer synthesis on the basis of solubility of all the monomers. Copper (II) sulfate and sodium ascorbate were chosen as catalysts, and polymerization proceeded at 50  C. To yield the final polymer 8, the Boc groups on monomer 4 were deprotected with trifluoroacetic acid in dichloromethane after polymerization (Fig. 1). To verify whether monomer 6 was successfully introduced into RCP, polymer formed by monomer 2 and monomer 4 was also synthesized and characterized by 1H NMR (Fig. 2B). Comparing these two 1H NMR spectra, the extra characteristic signals of monomer 6 were found in 1H NMR spectrum of polymer 8 (Fig. 2C), which indicated that the disulfide bone in monomer 6 was successfully introduced into RCP. The molar ratio of disulfide bond to triazole groups determined from the relative peak area of methylene groups adjacent to disulfide bond to triazole groups was about 15%. The relative weight

Fig. 2. 1H NMR spectra of monomer 6 (A), non-reduction-sensitive click polymer synthesized from monomer 2 and 4 (B), and reduction-sensitive click polymer synthesized from monomer 2, 4, and 6 (C). The red arrows indicated the signals assigned to peaks of monomer 6.

molecular weight of RCP was 1.1  104 Da, and the polydispersity (Mw/Mn) of RCP was 1.2 measured by GPC, which demonstrated that click polymers could be synthesized by more than two monomers using “one-pot synthesis” strategy. 3.2. Characteristics and reduction-sensitivity of RCPNs The ability of RCP to condense DNA was evaluated by particle size and z potential measurements. As shown in Fig. 3A, RCP could efficiently condense DNA into nanoparticles with average sizes about 190 nm at N/P ratio over 1:1. The particle size decreased with N/P ratio increasing, and the smallest size (151.9  4.6 nm) was observed for the RCPNs at N/P ratio 32. The z potential of RCPNs increased from 0.34 to 20.24 mV when the N/P ratio increased from 1:1 to 64:1. The morphology of RCPNs at N/P ratio 32 was evaluated by AFM. From AFM image, the particles were round and uniform with size about 100 nm (Fig. 3B). Because AFM measurement requires sample to air-dry before analysis, the sizes of particles measured by AFM are always smaller than the sizes measured by particle sizing system with samples suspended in solution. The formation of polymer/DNA complex nanoparticles was also confirmed by gel retardation assay. Fig. 4A showed gel electrophoresis of RCPNs with different N/P ratios without DTT. The

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electrophoretic mobility of DNA could be completely retarded at N/ P ratio 1, which indicated the strong binding capacity of RCP and DNA. It is reported that the disulfide bond could be cleaved inside the cell because of the relatively high concentration of GSH, and treatment of the complex nanoparticles with a strong reducing agent DTT (2.5 mM) could mimic the reductive environment inside cells to bioreductively cleave the disulfide bond [25,26]. Therefore, DTT was chosen to reduce disulfide bond in RCP and evaluate the reduction-sensitivity of RCPNs in this work. After incubation with 2.5 mM DTT for 30 min, RCP was unable to condense DNA. Free DNA strips appeared in agarose gel at all lanes loaded with RCPNs at different N/P ratios (Fig. 4B). The lack of DNA condensing ability after treated with DTT could also be observed from particle size analysis. The particle sizes of RCPNs increased sharply after incubation with 2.5 mM DTT. The particle size of RCPNs at N/P ratio 32 increased from 151.9  4.6 nm to 641.3  39.6 nm after incubated with DTT for 10 min (Fig. 4C), and the particle size could not be detected when extended the incubation time. The reductionsensitivity of RCPNs attributes to the cleavage of disulfide bond in RCP backbone, which could be confirmed by GPC analysis (Fig. 4D). After DTT treatment, the disulfide bond cleaved, and the click polymer could degrade into oligomers with smaller molecular weights. 3.3. In vitro transfection and cytotoxicity of RCPNs In order to evaluate the transfection capability of RCPNs, in vitro transfection experiments of RCPNs were assessed in MCF-7 and MCF-7/ADR cells, respectively (Fig. 5A). The transfection efficiency of RCPNs loading plasmid EGFP-N1 as reporter gene was measured by flow cytometry. On both cell lines, the transfection efficiency of RCPNs increased with N/P ratio from 8 to 32, and the best transfection efficiency of RCPNs against MCF-7 and MCF-7/ADR cells was obtained at N/P ratio 32, which was statistically higher than that of PEINs at mass ratio 3, which is optimal for gene transfection of PEINs in this study. The cytotoxicity profiles of RCPNs were shown in Fig. 5B and C. RCPNs showed essentially low cytotoxicity in both MCF-7 and MCF7/ADR cells with cell viability over 60% even at polymer concentration of 200 mg/ml for 24 h incubation. On contrast, PEINs at mass ratio 3 showed severe toxicity with either 2 h or 24 h incubation.

The low cytotoxicity of RCPNs could attribute to the bioreducible disulfide bonds in the main chain of RCP, which could be rapidly degraded by GSH within the cells. According to the above results, reduction-sensitive click polymer synthesized in this work could be used as an efficient and safe gene carrier. 3.4. P-gp inhibition To investigate the capability of RCPNs loading plasmid iMDR1pDNA to inhibit P-gp expression on drug resistance cells, the expression of P-gp on MCF-7 and MCF-7/ADR cell membrane was first determined by flow cytometry using fluorescence-labeled anti-MDR1 antibody. The fluorescent intensity of MCF-7 cells labeled by the specific antibody was similar to that labeled by the isotype control, and the P-gp positive cells were scarcely found. On the contrary, the drug-resistant MCF-7/ADR exhibited a strong fluorescent peak when labeled with anti-MDR1 antibody with 56.46% positive cells compared with that labeled by the isotype control. These results demonstrated the overexpression of P-gp in the resistant MCF-7/ADR cells. To validate the effectiveness of plasmid iMDR1-pDNA that could specifically silence MDR-1 to inhibit P-gp expression as previously reported, P-gp expression on both cell lines was determined after transfected RCPNs loading plasmid iMDR1-pDNA for 48 h (Fig. 6A and B). As expected, RCPNs could significantly inhibit P-gp expression in MCF-7/ADR cells with the fluorescent peak in the histogram corresponding to P-gp significantly decreased. The P-gp positive cells decreased from 56.46% to 12.87%. On the contrary, no obvious difference was observed in the histogram of MCF-7 cells before and after treated with RCPNs. The inhibitive effect of RCPNs on P-gp in MCF-7/ADR cells was also validated by western blot analysis. As showed in Fig. 6C, there was a significant decrease of P-gp in RCPNs treated cells compared with the untreated cells. These results indicated that RCPNs loading plasmid iMDR1-pDNA could specifically silence MDR-1 to inhibit P-gp expression in drug-resistant MCF-7/ADR cells. 3.5. Enhancement of chemosensitivity in treated drug-resistant tumor cells The sensitivity of MCF-7/ADR to ADR was first compared with drug sensitive MCF-7 cells. The IC50 value of ADR for MCF-7 cells

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Fig. 4. Evaluation of reduction-sensitivity of RCP and RCPNs. (A) Agarose gel electrophoresis of RCPNs at various N/P ratios. Lane 1: naked DNA control; Lane 2 to 7: complexes prepared at N/P ratios of 1, 2, 4, 8, 16, 32, and 64 in water, respectively. (B) Agarose gel electrophoresis of RCPNs after treated with 2.5 mM DTT. (C) Mean particle sizes of RCPNs at N/P ratio 32 before and after treated with DTT. (D) GPC spectra of RCP before and after treated with DTT.

were 0.85 mg/ml, however, over 50% of MCF-7/ADR cells survived after incubation with 0.1 mg/ml ADR for 24 h. One of the reasons for the resistance of MCF-7/ADR to ADR could be the P-gp mediating efflux. From in vitro cellular uptake, it could be found that the cellular uptake of ADR in MCF-7/ADR cells was significantly lower than in MCF-7 cells under the same concentration (data not shown). The overexpression of P-gp in MCF-7/ADR cells led to efflux of ADR and a reduction of ADR accumulation, which resulted in weakened cytotoxicity of ADR to MCF-7/ADR cells. To examine whether the stable expression of MDR-1 shRNA could inhibit the function of P-gp and enhance the chemosensitivity of MCF-7/ADR to ADR, we performed in vitro cellular uptake and cytotoxicity experiments. As shown in Fig. 7A, after transfection of RCPNs, the cellular uptake of ADR significantly increased with mean fluorescent intensity increasing from 37.4 to 82.9 (p < 0.01). As control, the mean fluorescent intensities of RCP/ neg-DNA complex nanoparticles and RCPNs both were neglectable, which indicated that the increased fluorescent intensity was due to the increased ADR accumulation rather than the material RCP or complex nanoparticles. The increased accumulation of ADR also could be observed under confocal microscope (Fig. 7B and C). The increased cellular uptake of ADR could significantly increase the cytotoxicity of ADR. As shown in Fig. 8, ADR revealed dose-

dependent cytotoxicity against MCF-7/ADR cells, and RCPNs at N/P ratio 32 showed neglectable cytotoxicity, but the combination of RCPNs and ADR could significantly increase ADR cytotoxicity. Combination of RCP/neg-DNA and ADR showed almost the same toxicity as ADR alone, which indicated that the increased ADR cytotoxicity could result from the expression of MDR-1 shRNA which inhibited the function of P-gp. These results demonstrated that RCPNs loading iMDR1-pDNA could inhibit P-gp expression and enhance the chemosensitivity of MCF-7/ADR cells to ADR. 3.6. In vivo therapeutic experiment Anti-tumor activity of combination of RCPNs and ADR was evaluated on drug-resistant tumor xenografts. The overexpression of P-gp on the membrane of cells extracted from tumor tissue was confirmed prior to the therapeutic experiment. For in vivo studies, lethal toxicity of RCPNs was first investigated in MCF-7/ADR breast tumor-bearing mice. No lethal toxicity was observed for RCPNs at N/ P ratio 32 at a dose of 3 mg DNA/kg. Therefore, under the experimental dose level (2 mg DNA/kg), RCPNs should not induce obvious toxicity in mice. Due to the severe toxicity of ADR, a dose of 8 mg/kg ADR which was well-accepted in mice was used [27], and a schedule of multiple dosing was performed to enhance the efficacy without

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increasing the acute toxicity. Animals were injected on day 0, 7 and 14. The effect of ADR and/or RCPNs treatment on tumor growth was presented in Fig. 9. RCPNs treatment did not show any inhibition effect on the growth of the MCF-7/ADR tumor. The relative tumor volume (RTV) of RCPNs group after 21 days from the first injection (Day 0) was 15.9  2.7, which showed no difference with that of the saline group (RTV ¼ 18.2  3.2), and indicated that only inhibition of P-gp had no effect on the tumor growth. ADR treatment could inhibit the growth of the drug-resistant tumor with RTV ¼ 7.1  1.9. The RTV of combination of RCPNs and ADR group was only 3.3  0.8, which indicated that the volume of tumors treated with combination of RCPNs and ADR was approximately a half of those treated with ADR only. These results demonstrated that RCPNs could effectively transfect DNA to express MDR-1 shRNA and inhibit the expression of P-gp in tumor cells, which led to sensitize tumor cells to ADR. In addition, there was no serious body weight loss in mice treated with RCPNs (data not shown), which demonstrated that the toxicity of RCPNs in mice was little, and RCPNs could be a safe gene delivery system. 4. Discussion

Fig. 5. In vitro transfection efficiency and cytotoxicity of RCPNs against MCF-7 and MCF-7/ADR cells. (A) The influence of the N/P ratio of RCPNs on the transfection efficiency. *p < 0.05 compared with PEINs. (B) Viability of MCF-7 cells when coincubated with RCPNs or PEINs for 2 h or 24 h with different polymer concentrations. (C) Viability of MCF-7/ADR cells when co-incubated with RCPNs or PEINs for 2 h or 24 h with different polymer concentrations.

In polymer-based gene delivery vectors, disulfide linkage in polymer backbone could be used to solve the contradiction between DNA condensation efficiency and the easy dissociation of DNA from complex nanoparticles in nucleus, and the contradiction between transfection efficiency and polymer toxicity since disulfide bond could be rapidly cleaved in the presence of high concentrations of glutathione (GSH) in intracellular environment. Previous reports about synthesis of polymers containing disulfide bond mainly focused on the conjugation of low molecular weight PEI into degradable PEI [28,29], modification of PEG in the side chains of polymers through disulfide bond [30], or preparation of poly(amido amine)s containing disulfide bond by Micheal addition [31,32]. However, there is no report about the synthesis of the linear cationic click polymer containing disulfide bond until today. “Click chemistry”, introduced by Sharpless et al. in 2001, has attracted much attention in synthetic chemistry. The copper(Ⅰ)-catalyzed azideealkyne cycloaddition, which is regarded as a prime example of “click chemistry”, has been used in various scientific disciplines such as drug discovery, bioconjugate synthesis, drug delivery and diagnostics owing to its convenient, quick, regioselective and chemoselective reaction [33,34]. The linear click polymer could be obtained by copolymerization of azide-functionalized monomer with alkyne-functionalized monomer. In previous reports, the syntheses of linear click polymers were all based on one or two kinds of monomers. In this work, we used three different monomers functionalized with either azide or alkyne to synthesize click polymer containing disulfide bond by a one-step route with more quickly and convenience. The newly synthesized RCP showed good transfection efficiency as gene delivery vector, which could be due to the following reasons. Firstly, the secondary amine in the polymer backbone could effectively interact with negatively charged DNA and condense DNA into round and uniform nanoparticles around 150 nm. Secondly, the amideetriazole moiety, which was formed during polymerization, could act as H-bond acceptors to increase hydrophobic interactions of the polymer with DNA, thus improve the polymereDNA binding stability and reduce the effects of serum during transfection [18,20,21]. Thirdly, the vinyl ether structure in the polymer could reduce polymer charge density and make polymer with more hydrophilicity. Polymer with low charge density could decrease the interaction between positive polymer and negative cell membrane to reduce cytotoxicity [35]. Finally, the disulfide bond in the polymer backbone could be degraded by GSH in intracellular environment to

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Fig. 6. RCPNs inhibition of P-gp expression of MCF-7/ADR cells. Flow cytometry analysis of P-gp expression on MCF-7/ADR (A) or MCF-7 (B) cell membrane before (1) or after (2) treated with RCPNs at N/P ratio 32. (C) Western blot analysis of P-gp down-regulation in MCF-7/ADR and MCF-7 cells. Lanes 1 and 2 represent untreated control and RCPNs treated cells.

improve DNA dissociation from complexes, thus to increase transfection efficiency, and at the same time, increase the degradability of polymer, thus to decrease cytotoxicity further. From in vitro transfection efficiency and cytotoxicity evaluation, it could be concluded that RCPNs could be a promising safe and efficient carrier for gene delivery. P-gp has been considered to be one of the most important targets to overcome MDR. It is estimated that about 40e50% of breast cancer patients demonstrate P-gp overexpression [6]. Therefore, the inhibition of the function of P-gp could be one of the effective ways to re-sensitize tumor cells to anti-cancer drugs during breast cancer chemotherapy. To inhibit the function of P-gp,

RNAi could be used to knockdown the expression of MDR-1 gene by an efficient and specific way. Instead of chemical synthetic siRNA or dsRNA, shRNA expression vector iMDR1-pDNA was constructed and used for MDR-1 gene silencing in this work. The plasmid vector could overcome the disadvantages of non-specificity related to introduction of long dsRNA and transient activity related to direct transfection of synthesized siRNA into cells [36,37]. From in vitro results of P-gp determination and sensitivity of MCF-7/ADR cells to ADR, it could be found that RCPNs loading iMDR1-pDNA could effectively deliver plasmid into cells to express shRNA and inhibit the P-gp expression (Fig. 6). The weakened drug efflux could significantly increase ADR accumulation (Fig. 7) and markedly

Fig. 7. (A) Flow cytometry analysis of cellular uptake of ADR in MCF-7/ADR cells treated with RCPNs at N/P ratio 32. Cells were incubated with 1 mg/mL drug for 2 h. **p < 0.01. (B) Confocal images MCF-7/ADR cells treated with RCPNs at N/P ratio 32 or not treated with RCPNs at N/P ratio 32 (C) before incubation with 1 mg/mL ADR for 2 h. Scale bar ¼ 50 mm.

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RCPNs ADR RCP/neg-DNA+ADR RCPNs+ADR

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Fig. 8. Cytotoxicity of free ADR against MCF-7/ADR cells after treated with RCPNs at N/P ratio 32. Cells were incubated with different drug concentrations for 48 h. *p < 0.05 and ** p < 0.01 compared with that of group treated with ADR only.

enhance cytotoxicity of ADR against MCF-7/ADR cells (Fig. 8). These results demonstrated the effectiveness of RCPNs as gene delivery vector and anti-MDR1 shRNA expression system. To estimate the effect of RCPNs to reverse MDR in vivo, MCF-7/ ADR breast tumor model was established. It was reported that shRNA-expressing plasmid target MDR-1 gene achieved good results in reversing MDR in stomach or liver tumor [38,39]. But no report was found to reverse MDR by inhibit MDR-1 gene in MCF-7/ADR breast tumor model. RCPNs loading iMDR1-pDNA could significantly suppress P-gp expression in tumor tissue and decrease drug efflux in tumor cells, thus enhance anti-tumor effects of ADR. The effective delivery of iMDR1-pDNA condensed by RCP into tumor tissue could attribute to the strong DNA binding ability of RCP to increase the

stability of RCPNs in vivo and the passive targeting ability of nanoparticles to tumor tissues via the EPR effect [40]. 5. Conclusion A reduction-sensitive linear cationic click polymer was synthesized via “click chemistry”. RCP could efficiently condense pDNA into nanoparticles, and RCPNs could dissociate in the presence of DTT due to the cleavage of disulfide bonds, which indicated the efficient DNA release under the reduction condition such as cytoplasm. In vitro transfection and cytotoxicity against MCF-7 and MCF-7/ADR cells showed that RCPNs could bring about higher transfection efficiency with much lower cytotoxicity than PEINs. RCPNs loaded with plasmid iMDR1-pDNA could inhibit P-gp expression, increase ADR accumulation and enhance cytotoxicity of ADR in MCF-7/ADR cells and anti-tumor effect on mouse xenograft model bearing ADR resistant human breast cancer. These results demonstrated that this RCP could be used as a potential safe and efficient non-viral vector for reversing MDR. Acknowledgement The National Basic Research Program of China (2010CB934000 and 2007CB935800）, the National Natural Science Foundation of China (30925041, 30873169), National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (No 2009ZX09501-024 and 2009ZX09301-001), and Shanghai Nanomedicine Program (1052nm06300) are gratefully acknowledged for financial support. Appendix

Fig. 9. Anti-tumor effects of RCPNs, ADR or the combination of RCPNs and ADR on nude mice bearing MCF-7/ADR. Data were given as mean  SD (n ¼ 6). *p < 0.05 and ** p < 0.01 compared with ADR group.

Figures with essential color discrimination. Figs. 2e4, 6, 7 and 9 in this article are difficult to interpret in black and white. The full color images can be found in the online version, at doi:10.1016/j. biomaterials.2010.11.001.

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