Magnetic DNA Vector Constructed from PDMAEMA Polycation and

Article pubs.acs.org/Langmuir

Magnetic DNA Vector Constructed from PDMAEMA Polycation and PEGylated Brush-Type Polyanion with Cross-Linkable Shell Ying Hao,† Mingzu Zhang,† Jinlin He,† and Peihong Ni*,†,‡ †

College of Chemistry, Chemical Engineering and Materials Science, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Soochow University, Suzhou 215123, China ‡ Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123, China S Supporting Information *

ABSTRACT: A novel magnetic-responsive complex composed of polycation, DNA, and polyanion has been constructed via electrostatic interaction. The magnetic nanoparticles (MNPs) were first coated with a polycation, poly[2(dimethylamino)ethyl methacrylate] end-capped with cholesterol moiety (Chol-PDMAEMA30), and then binded with DNA through electrostatic interaction; the complexes were further interacted with the brush-type polyanion, namely poly[poly(ethylene glycol)methyl ether methacrylate]-blockpoly[methacrylic acid carrying partial mercapto groups] (PPEGMA-b-PMAASH). The resulting magnetic particle/ DNA/polyion complexes could be stabilized by oxidizing the mercapto groups to form cross-linking shell with bridging disulfide (S−S) between PPEGMA-b-PMAASH molecular chains. The interactions among DNA, Chol-PDMAEMA coated MNPs, and PPEGMA-b-PMAASH were studied by agarose gel retardation assay. The complexes were fully characterized by means of zeta potential, transmission electron microscopy (TEM), dynamic light scattering (DLS) measurements, cytotoxicity assay, antinonspecific protein adsorption, and in vitro transfection tests. All these results indicate that this kind of magneticresponsive complex has potential applications for gene vector.

1. INTRODUCTION Gene therapy has been known as a promising treatment for numerous hard curable diseases,1 and its safety and efficiency play the most challenging role. It has been generally acknowledged that nonviral gene delivery systems are safer than viral vectors; however, their relatively low transfection efficiency limited their practical applications.2−4 A great number of efforts have been made to increase the transfection efficiency of nonviral gene vectors,2 among which introduction of magnetic nanoparticles is an alternative because they can provide target effect for the vectors.5 According to the previous literature, the use of magnetic nanoparticles for in vitro and in vivo transfection of recombinant adeno-associated virus 2 (rAAV) by linking viral vectors to magnetic microspheres was first reported by Mah et al. in 2000.6,7 Because of these pioneering studies, magnetic nanoparticles have drawn much attention due to their ability to be manipulated externally and enhancement of the uptake in biomedical fields.8−12 However, bare magnetic nanoparticles often tend to aggregate in tissue fluids. It is necessary to improve the water dispersity and biocompatibility of magnetic nanoparticles. A majority of approaches have been focused on coating iron oxide nanoparticles with water-soluble cationic polymer such as polyethylenimine (PEI), poly(L-lysine) (PLL), and poly[2(dimethylamino)ethyl methacrylate] (PDMAEMA).13−15 © 2012 American Chemical Society

Cationic polymers exhibit efficient DNA condensation, but their high positive charge density lead to high cytotoxicity.16−18 Some of cationic charges can be partially masked by DNA condensation,19 but unspecific interactions and nonspecific adsorptive endocytosis will take place, resulting in the aggregations formed with blood cells and plasma proteins after intravenous injection of cationic gene vectors.20−22 An effective approach to overcome the cytotoxicity of polycation is to incorporate them with nonionic hydrophilic units to shield the positive charges, such as poly(ethylene glycol) (PEG) and poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA).23,24 As a well-known nontoxic and anti-immunogenic polymer, PEG is usually used to prolong the systemic circulation time and decrease the cytotoxicity of gene carriers, which is possibly due to the highly flexible PEG chains acting as a molecular brush to sterically interfere with nonspecific interactions between the serum-driven components and carriers.25 Moreover, PEGylation can also provide stabilization by decreasing interparticular aggregation and enhancing the solubility of polyplexes in aqueous solution.2,26 Maruyama et al.27 reported polymer brush effect of polysaccharide graft chains, which Received: January 14, 2012 Revised: March 24, 2012 Published: March 26, 2012 6448

dx.doi.org/10.1021/la300208n | Langmuir 2012, 28, 6448−6460

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Scheme 1. Schematic Diagram of the Magnetic-Responsive Complexes Fabricated by the Brush-Type Copolymer Carrying Mercapto Groups

under cellular reducing environment and the encapsulated DNA was released. Very recently, we introduced a magnetic core in this kind of gene delivery complex,41 but the biomedical tests were less investigated. Herein, considering the advantages of PEGylated brush-type polyanion and targeting magnetic nanoparticles, a new magnetic-responsive complex with disulfide bridge linking shell and brush-type PEG on the periphery was constructed. Two improvements were made here compared with our previous works: (i) a new strategy was adopted in the preparation of polyanion with partial mercapto groups in order to increase the stability of this polymer during formulation; (ii) the PEGylated brush-type polyanion increase the antinonspecific protein adsorption and the preliminary in vitro transfection was studied. The brush-type polyanion PPEGMA-b-PMAASH was synthesized via three steps: (1) preparation of PPEGMA-b-PtBMA diblock copolymer via ATRP; (2) hydrolysis of poly(tert-butyl methacrylate) in PPEGMA-b-PtBMA to yield PPEGMA-b-PMAA; (3) replacement of partial carboxyl groups (−COOH) in PPEGMA-bPMAA by mercapto groups (−SH) and yielding PPEGMA-bPMAASH. This polyanion was used to bind the polycation Chol-PDMAEMA coated magnetic nanoparticles (MNPs)/ DNA complexes by electrostatic interaction, as shown in Scheme 1. The stable complexes were formed by oxidizing the mercapto groups using hydrogen peroxide or oxygen to generate disulfide linkages (S−S) between PPEGMA-bPMAASH. The carried DNA will be released once the disulfide linkages were cleaved in the cellular reducing environment. We also studied the properties of these complexes by using zeta potential, transmission electron microscopy (TEM), dynamic light scattering (DLS) measurements, cytotoxicity assay,

exhibited resistance against self-aggregation and nonspecific adsorption of serum proteins. They also studied a series of cationic comb-type copolymers with highly dense water-soluble side chains and found that these polyplexes stabilized by brush polymer showed longer circulation time in mouse bloodstream.28 Therefore, the dense polymer brushes may shield positive charges of cationic copolymers and more resistant to nonspecific adsorption.2,29 Poly(ethylene glycol) monomethacrylate (PEGMA) is a useful macromonomer bearing short oligo(ethylene glycol) chains. Well-defined brush-type polymer PPEGMA can be prepared via controlled radical polymerization techniques such as atom transfer radical polymerization (ATRP)29−31 or reversible addition−fragmentation chain transfer (RAFT).32,33 In the past decade, brush-type copolymers based on PEGMA have been of growing interest in biomedical applications because of their unique properties such as excellent bioinert properties and tunable thermoresponsiveness.34−36 For instance, a series of tailor-made copolymers of DMAEMA and OEGMA were synthesized and used as nonviral gene transfer agents. The results indicated that these copolymers exhibited excellent colloidal stability, low cytotoxicity, and improved in vivo gene expression.37,38 Moreover, the temperature-responsive PEGMA-based nanocapsules have potential applications in drug delivery.39 We have also noted that few studies have exploited the magnetic responsive gene vectors fabricated by brush-type copolymer. Our group designed and fabricated a gene carrier model by means of electrostatic interactions of polycation, DNA, and linear polyanion.40 These complexes were stabilized by oxidizing the mercapto groups on the polyanion chains and forming bridging disulfide linkages, which could be broken 6449


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CH2Cl2/DMF (3:1 v/v, 12 mL). The mixture was stirred at 0 °C, and a solution of N,N′-dicyclohexylcarbodiimide (DCC, 0.085 g, 0.41 mmol) in 4 mL of CH2Cl2 was added dropwise over 30 min. The reaction was then stirred for 12 h under nitrogen at room temperature. The insoluble solid was removed by filtration, and the filtrate was concentrated and precipitated into 50 mL of cold ethyl ether. The precipitate was filtered and dried in a vacuum oven at room temperature to yield PPEGMA-b-PMAAS‑S‑NH‑Boc. Next, the PPEGMA-b-PMAAS‑S‑NH‑Boc was dissolved in PBS buffer (pH 7.4, 10 mM), and DTT (10 mM) was added to cleave the disulfide linkages for 12 h. The mixture was subsequently purified by dialysis with deionized water (MWCO 3500) for 72 h, followed by freeze-drying to yield a white solid as the product PPEGMA-b-PMAASH. 2.3. Preparation of Polycation Coated Magnetic Nanoparticles (MNPs). In order to prepare a polycation, cholesterol-ended poly[2-(dimethyl)ethyl methacrylate] (Chol-PDMAEMA30) was synthesized by oxyanion-initiated polymerization as reported in our previous work.40 Oleic acid (OA) coated iron oxide nanoparticles were prepared by the coprecipitation method.42 OA coated magnetic nanoparticles (10 mg) and Chol-PDMAEMA (100 mg) were dissolved in 10 mL of chloroform, and a ligand exchange reaction was carried out at 20 °C for 72 h. After that, the mixture was concentrated, precipitated in hexane, and dried under vacuum. The dried magnetic nanoparticles were then purified by repeated washing with deionized water and magnetic separation to remove the free polymer. 2.4. Preparation of Brush-Type Polyanion/Polycation Coated MNPs/DNA Ternary Complexes. Three solutions of DNA, Chol-PDMAEMA coated MNPs, and polyanion were separately prepared by dissolving the corresponding samples in PBS buffer (pH 7.4, 10 mM) with the concentration of 1 mg/mL. DNA solution was filtered through 0.45 μm filter (Shanghai CAIENFU Technology). The polycation solution was added to DNA solution to form complexes with various mass ratios of [Chol-PDMAEMA coated MNPs]/[DNA] and then mixed for 20 s using a vortex mixer. Subsequently, the polyanion solution of PPEGMA-b-PMAASH in PBS buffer was added to the Chol-PDMAEMA coated MNPs/DNA complex system with different mass ratios of [MAA]/[CholPDMAEMA coated MNPs]. Hydrogen peroxide solution (H2O2, 10 mM) was then added to stabilize the complexes by oxidizing the mercapto groups and forming disulfide linkages (S−S) between the PPEGMA-b-PMAASH chains. For the in vitro degradation analysis, DTT (10 mM) was added into the complex solution to destroy the disulfide linkages and release DNA. 2.5. Materials Characterizations. The chemical structures of PPEGMA, PPEGMA-b-PtBMA, PPEGMA-b-PMAA, PPEGMA-bPMAAS−S−NH‑Boc, and PPEGMA-b-PMAASH were determined with a 400 MHz 1H NMR instrument (INOVA-400), using CDCl3 or DMSO-d6 as solvent. GPC measurements for PPEGMA and PPEGMA-b-PtBMA were carried out at 30 °C using a Waters 1515 gel permeation chromatograph (GPC) instrument with a PLgel 5.0 μm bead size guard column (50 × 7.5 mm2), followed by two linear PLgel columns (500 Å and Mixed-C) and a differential refractive index detector. THF was used as the eluent at a flow rate of 1.0 mL/min, and calibration was performed with polystyrene standards. 2.6. Cell Toxicity Test by MTT Assay. A standard MTT assay was employed to determine the cytotoxicity of Chol-PDMAEMA coated magnetic nanoparticles and complexes against human embryonic kidney cells (HEK 293T cells) and human cervix carcinoma cells (HeLa cells). Cells were cultured in growth medium (DMEM for HEK 293T cells, RPMI-1640 for HeLa cells) containing 10% heatinactivated fetal bovine serum (FBS), 1% penicillin, and streptomycin, at 37 °C in 5% CO2 atmosphere. First, cells were seeded in a 96-well plate at a density of about 5 × 104 cells/well and incubated for 24 h in 100 μL of growth medium/well containing 10% serum. A series of polymer and polycation coated MNPs solutions with different concentrations were individually added into different wells and incubated with cells for 48 h. The standard MTT assay was carried out to determine the cell viability relative to the control untreated cells on a microplate reader (PowerWave XS, Bio-Tek). The cell viable rate was calculated by the equation of ODtest/ODcontrol × 100%, in which

antinonspecific protein adsorption, and in vitro transfection tests.

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(ethylene glycol)methyl ether methacrylate (PEGMA, M̅ n = 300 g/mol, Aldrich) was dissolved in THF and passed through a neutral Al2O3 column, concentrated in a rotary evaporator, and finally dried under reduced pressure. Cystamine dihydrochloride (TCI), tert-butyl methacrylate (tBMA, TCI), methyl-2-bromopropionate (MBP, Aldrich), copper(I) chloride (CuCl, 99.999%, Alfa Aser), dithiothreitol (DTT, Alfa Aesar), and methylthiazolyltetrazolium (MTT, Sigma-Aldrich) were used as received. 4-(Dimethylamino)pyridine (DMAP) and N,N′-dicyclohexylcarbodiimide (DCC) were purchased from Shanghai Medpep Co. Calf thymus DNA (Aldrich) and Plasmid pUC18 DNA (Takara) were used for agarose gel electrophoresis, zeta potential, DLS, and TEM measurements. 2,2Bipyridyl (bpy), trifluoroacetic acid (TFA), di-tert-butyl dicarbonate (Boc2O), anhydrous ethanol, hexane, and other reagents were purchased from Sinopharm Chemical Reagent Co. and used as received. Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) was obtained from Shanghai Ebioeasy Co. 2.2. Synthesis of Brush-Type Diblock Copolymer PPEGMAb-PMAASH. 2.2.1. Preparation of PPEGMA via ATRP Using MBP as the Initiator. MBP (50 μL, 0.44 mmol), PEGMA (4.0 g, 13.34 mmol), CuCl (44.0 mg, 0.44 mmol), and bpy (138.9 mg, 0.88 mmol) were added into a 50 mL dry flask containing 8.75 mL of solvent (ethanol/water, 95/5, v/v). The reaction mixture was purged with argon for 10 min, and the flask was then sealed. The polymerization was carried out at 30 °C under stirring, and the reaction was quenched after 12 h by exposing the mixture to air. The reaction mixture was diluted with ethanol and passed through a neutral Al2O3 column to remove the catalyst. The solution was concentrated and precipitated in cold hexane for three times; the brush-type polymers of poly(ethylene glycol)methyl ether methacrylate (PPEGMA) with chlorine group in the end of chain were then collected and dried in vacuum at room temperature for 24 h. The PEGMA monomer conversion was about 70%, and M̅ n of PPEGMA was 9800 g/ mol as determined by GPC analysis. 2.2.2. Preparation of PPEGMA-b-PtBMA Using PPEGMA as the Macroinitiator. The synthesis procedure for the brush-type diblock copolymer PPEGMA-b-PtBMA was similar to the above-mentioned method for the ATRP of PEGMA. PPEGMA (2.0 g, 0.204 mmol), tBMA (2.3 g, 16.32 mmol), CuCl (20.2 mg, 0.204 mmol), and bpy (63.7 mg, 0.408 mmol) were dissolved in 18.8 mL of ethanol/water mixture solvent (95/5, v/v). The solution was purged with argon for 10 min, and the flask was sealed. The polymerization was carried out at 30 °C for 24 h. After that, the reaction was quenched by exposing the mixture to air. The reaction mixture was passed through a neutral Al2O3 column to remove the catalyst, concentrated, and precipitated in cold hexane for three times. The brush-type diblock copolymer was then obtained as a viscous solid after drying in vacuum at room temperature for 48 h. The monomer conversion was about 50%, and M̅ n of PPEGMA-b-PtBMA was 17280 g/mol obtained from GPC analysis. 2.2.3. Synthesis of PPEGMA-b-PMAA via Hydrolysis of PPEGMAb-PtBMA. A typical hydrolysis procedure was described as follows: To a solution of PPEGMA-b-PtBMA (1.0 g, 0.058 mmol) in 10 mL of CH2Cl2 in a 100 mL round-bottom flask, excess trifluoroacetic acid (TFA, 1.23 mL, 16.6 mmol) was slowly added at 0 °C under stirring. After the addition was completed, the reaction was kept at room temperature for 55 h. The reaction mixture was concentrated and precipitated into a mixture of cold hexanes and ethyl ether (10/1, v/v) for three times. The PPEGMA-b-PMAA was then collected and dried in vacuum at room temperature for 24 h as a white powder. 2.2.4. Synthesis of Diblock Copolymers PPEGMA-b-PMAASH Containing Mercapto Groups. To a 50 mL of round-bottom flask, PPEGMA-b-PMAA (0.5 g), monoboc-cystamine (0.05 g, 0.20 mmol, target modification = 20 mol %), and 4-(dimethylamino)pyridine (DMAP, 0.025 g, 0.20 mmol) were dissolved in a mixed solvent of 6450


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Scheme 2. Synthesis of PPEGMA-b-PMAASH via Three Steps: (a) Synthesis of Brush-Type Copolymer PPEGMA-b-PtBMA via ATRP; (b) Hydrolysis of PPEGMA-b-PtBMA Using TFA Treatment; (c) Partial Mercapto Substitution of Carboxyl Groups in PPEGMA-b-PMAA Brush-Type Copolymer

ODtest and ODcontrol are the absorbance values of the testing well (in the presence of samples) and the control well (in the absence of samples), respectively. 2.7. Biophysical Properties Measurement. Transmission electron microscopy (TEM) images were obtained from the instrument (TECNAI G2 20, FEI Co.) at an acceleration voltage of 200 kV. The polymer solutions were prepared by dissolution of the copolymer in 10 mM PBS buffer solution. The carbon coated copper grid (400 mesh) was immersed in the aqueous polymer solution, taken out, and dried at room temperature for 1 day prior to the measurement. In addition, the size and zeta potential of the nanoparticles in aqueous solution were measured by a Zetasizer Nano ZS (Malvern Instruments Ltd.). All measurements were carried out at 25 °C, and the data were analyzed with Zetasizer software (DTS). 2.8. Agarose Gel Electrophoresis. To evaluate DNA condensation ability of the polymers, the complex solution with different mass ratios of [Chol-PDMAEMA coated MNPs]/[DNA] ranging from 0 to 10, mixed with 2 μL of loading buffer (85% glycerol and 15% bromophenol blue), was run on a 0.8 wt % agarose gel with ethidium bromide (0.5 μg/mL) staining in Tris-borate-EDTA buffer

(TBE: 40 mM trisborate, 1 mM EDTA, and pH 7.4). The retardation assays were performed at a voltage of 90 V for 30 min. The gel was visualized by a UV irradiation (M-15E, UVP Inc., Upland, CA) to show the location of DNA. 2.9. Anti-nonspecific Protein Adsorption Test. The brush-type copolymer PPEGMA-b-PMAA (50 mg) was dissolved in 2 mL of THF. The solution was dropped on a glass slide and dried at room temperature for 48 h to volatilize the solvent and form the polymer film. The polymer film was then coated with FITC-BSA solution (6.7 mg/mL) and incubated statically at 37 °C for 30 min. The upper clear solution was removed, and the film was washed with distilled water to remove the unadsorbed FITC-BSA. At last, the glass slides were observed and photographed using the upright fluorescence microscope (Leica DM4000 M) with the Leica DFC420 C digital camera running at blue light excitation (490 nm excitation wavelength). 2.10. In Vitro Transfection. In vitro gene transfection was performed in HEK 293T cells as well as HeLa cells. Cells were cultured in a 24-well plate at the density of 2 × 105 cells/well and incubated for 24 h in 500 μL of growth medium containing 10% fetal calf serum (FBS) at 37 °C under 5% CO2 atmosphere. The PPEGMAb-PMAASH, Chol-PDMAEMA coated MNPs, and DNA encoding for 6451


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Figure 1. 1H NMR spectra of (A) PPEGMA homopolymer and (B) PPEGMA-b-PtBMA brush-type copolymer. enhanced green fluorescence protein (EGFP) solutions at different mass ratios mixed with the culture medium (Opti-MEM I reduced serum medium for HEK 293T cells and RPMI-1640 medium with or without 10% serum for HeLa cells). They were incubated for 20 min at room temperature to allow the formation of PPEGMA-b-PMAASH/ Chol-PDMAEMA coated MNPs/DNA complexes. Subsequently, 100 μL of the produced complexes was added to each well and mixed gently by rocking the plate back and forth and further incubating for 6 h at 37 °C under 5% CO2 atmosphere. After that, the complexes were removed, and 500 μL of fresh culture medium containing 10% serum was added. The cells were then incubated at 37 °C in a CO2 incubator for 48 h. At last, the gene transfection was characterized by fluorescence microscopy (BX60, Olympus, Japan), living cell imaging system (CELL’R, Olympus, Japan), and flow cytometry (FC500, Beckman).

Figure 2. GPC traces of PPEGMA24 homopolymer (M̅ n = 7330 g/ mol, M̅ w/M̅ n = 1.19), and PPEGMA24-b-PtBMA33 diblock copolymer (M̅ n = 11960 g/mol, M̅ w/M̅ n = 1.27).

3. RESULTS AND DISCUSSION 3.1. Synthesis of Brush-Type Diblock Copolymer PPEGMA-b-PMAASH. 3.1.1. Synthesis of PPEGMA-bPtBMA. To synthesize a diblock copolymer containing both PPEGMA and PtBMA blocks, we first prepared PPEGMA homopolymer via ATRP using methyl 2-bromopropionate (MBP) as the initiator in ethanol/water mixture solvent. The obtained chlorine-end-capped PPEGMA was then used as a macroinitiator to polymerize tBMA monomer, as shown in Scheme 2. The chemical structure, composition, and molecular weight of PPEGMA and PPEGMA-b-PtBMA were verified with 1 H NMR and GPC measurements. Figure 1 shows the 1H NMR spectra of PPEGMA homopolymer and PPEGMA-bPtBMA diblock copolymer. The characteristic chemical shifts are identified as follows: δ 0.9−1.1 ppm (signal a, H of −CH3 from PPEGMA), δ ∼ 1.8 ppm (signal b, H of −CH2− from PPEGMA), δ ∼1.4 ppm (signal i, H of −O−C(CH3)3 from PtBMA). GPC traces of PPEGMA24 and PPEGMA24-bPtBMA33 are representatively shown in Figure 2, from which one can find the increasing evolution of molecular weight from PPEGMA macroinitiator to the diblock copolymer PPEGMA-

b-PtBMA. The detailed number-average molecular weights (M̅ n) and the polydispersity indices (M̅ w/M̅ n) of samples are summarized in Table 1. Table 1. Characteristics of the Molecular Weights and Molecular Weight Distributions of PPEGMA macroinitiators and PPEGMA-b-PtBMA Diblock Copolymers sample 1 2 3 4 5 6 6452

polymer composition PPEGMA24 PPEGMA32 PPEGMA24-bPtBMA49 PPEGMA24-bPtBMA33 PPEGMA32-bPtBMA53 PPEGMA32-bPtBMA50

M̅ n (GPC) (g/mol)

M̅ w (GPC) (g/mol)

M̅ w/M̅ n

7 330 9 800 14 110

8 740 12 320 17 880

1.19 1.25 1.26

11 960

15 290

1.27

17 280

24 040

1.39

16 930

21 610

1.27


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Figure 3. 1H NMR spectrum of brush-type copolymer PPEGMA-b-PMAA after hydrolysis of PPEGMA-b-PtBMA copolymer.

Figure 4. 1H NMR spectra of brush-type copolymers for (A) PPEGMA-b-PMAAS‑S‑NH‑Boc and (B) PPEGMA-b-PMAASH.

3.1.2. Synthesis of PPEGMA-b-PMAA via the Hydrolysis of PPEGMA-b-PtBMA. To obtain the double hydrophilic diblock copolymer PPEGMA-b-PMAA, trifluoroacetic acid (TFA) was used to accelerate the hydrolysis of the tert-butyl groups form PtBMA segment because TFA does not affect other ester linkages in the polymer.43 The cleavage reaction was performed in dichloromethane with excessive TFA at room temperature for 55 h as shown in Scheme 2. In comparison of the 1H NMR spectrum in Figure 3 for PPEGMA-b-PMAA with that in to Figure 1B for PPEGMA-b-PtBMA, we can find that the chemical shift of the protons in tert-butyl groups at δ 1.4 ppm completely disappear, and a newly appeared chemical shift at δ 12.4 ppm can be ascribed to the proton of carboxyl groups in

PMAA block, confirming that PPEGMA-b-PMAA diblock copolymer had been successfully synthesized. Furthermore, in order to investigate the effect of TFA treatment on PPEGMA block, we carried out a control experiment using PPEGMA homopolymer under the same condition as that for PPEGMAb-PtBMA diblock copolymer. The 1H NMR spectra of PPEGMA homopolymer before and after TFA treatment are shown in Figure S1 of the Supporting Information, which indicated that TFA served for the cleavage of tert-butyl ester group had no apparent influence on PPEGMA block. 3.1.3. Synthesis of PPEGMA-b-PMAASH Diblock Copolymer Containing Mercapto Groups. In our previous work, we used cysteamine hydrochloride to react with polyanion; however, it 6453


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3.3. Agarose Gel Electrophoresis. The binding capacity with DNA of Chol-PDMAEMA coated MNPs was investigated by comparative agarose gel electrophoresis experiments. Figure 6

may result in the oxidization of mercapto groups during the preparation of product.40,41 Herein, mono-Boc-cystamine, which was prepared via the reaction of cystamine dihydrochloride with di-tert-butyl dicarbonate (Boc2O) according to the previous literature,44 was utilized to react with PPEGMA-bPMAA. The resulting PPEGMA-b-PMAAS‑S‑NH‑Boc was characterized by 1H NMR spectroscopy, as shown in Figure 4A. Subsequently, DTT was added to cleave disulfide linkages to get the polyanion with partial mercapto groups in the side chains of PMAA segment, that is, PPEGMA-b-PMAASH. As shown in Figure 4B, the complete disappearance of the chemical shift ascribed to tert-butyl on the Boc group at δ 1.4 ppm indicates the cleavage of disulfide linkages and provides evidence for the successful synthesis of PPEGMA-b-PMAASH brush-type copolymer. 3.2. Preparation of Polycation Coated MNPs. Polycation coated MNPs were prepared from oleic acid (OA) coated MNPs based on a ligand-exchange reaction with CholPDMAEMA, which was referred to the reaction of polyethylenimine (PEI) coated MNPs.45 The strong hydrophobic cholesterol groups of Chol-PDMAEMA have a highly affinity to oleic acid ligand on the surface of Fe3O4 nanoparticles, so that Chol-PDMAEMA could be adsorbed onto the surface of Fe3O4 MNPs to replace OA. We could obtained the well-dispersed and hydrophilic MNPs with Fe3O4 magnetic nanoparticles as core and hydrophilic PDMAEMA as corona, designated CholPDMAEMA coated MNPs or polycation coated MNPs. Figure 5 exhibits the optical photograph of polycation coated MNPs in

Figure 6. Gel retardation assay results for Chol-PDMAEMA coated MNPs/calf thymus DNA complexes at various mass ratios. Lane 1 is the DNA control; lanes 2−12 correspond to 0.2, 0.5, 0.7, 1.0, 1.5, 3.0, 3.5, 5.0, 7.0, 9.0, and 10.0 (mass ratios), respectively.

shows the agarose gel electrophoresis image of Chol-PDMAEMA coated MNPs/calf thymus DNA complexes at various mass ratios ranging from 0 to 10. DNA migration could be completely retarded when the [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio was above 7, which indicates that DNA was saturated with the Chol-PDMAEMA coated MNPs, and effective condensation can stabilize DNA against degradation by nuclease.46 Figure 7 shows the effect of PPEGMA-b-PMAASH (mercapto substitution product from sample 3 in Table 1) on the electrophoretic migration of DNA from Chol-PDMAEMA coated MNPs/calf thymus DNA complexes without and with addition of hydrogen peroxide (H2O2) solution. In the absence of H2O2, with the increasing mass ratio of [MAA]/[CholPDMAEMA coated MNPs], DNA migrated when the mass ratios is higher than 0.3 (lane 8), and it was completely released at the mass ratios from 0.8 to 1.5 (lanes 10−12), as shown in Figure 7A, due to the chain-exchange reaction reported in the previous paper.40 When H2O2 solution was added, disulfide linkages between PMAASH chains could generate and protected DNA from the chain-exchange reaction. As seen from Figure 7B, at the same mass ratio of [MAA]/[Chol-PDMAEMA coated MNPs], DNA was still fixed, and almost no release could be observed at the mass ratios from 0.8 to 1.5 (lanes 10− 12). It indicated that disulfide linkages can significantly weaken the effect of the chain-exchange reaction and stabilize the complexes. Figure 8 shows the in vitro degradation of disulfidecross-linked PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNA complexes in the presence of DTT. It can be observed that DNA began to migrate in lane 4 and could be completely released from lane 5 to lane 12, verifying the cleavage of disulfide linkages of complexes. 3.4. Cell Toxicity of Polycation Coated Magnetic Nanoparticles and Complexes. Cytotoxicity is one of the most important factors for gene vector and has a strong impact on gene transfection. Here, cell toxicity of cationic polymer Chol-PDMAEMA coated MNPs and complexes was evaluated by MTT, using commercial branched PEI (25 kDa) as a reference. Figure 9 shows the in vitro cytotoxicity of CholPDMAEMA coated MNPs and branched PEI (25 kDa) against HEK 293T cells and HeLa cells. The cell viabilities decreased

Figure 5. Optical photographs of Chol-PDMAEMA coated MNPs dispersed in deionized water (left) and being pulled by a magnet (right).

deionized water. It was found that the modified MNPs can be well dispersed in water, whereas aggregated in the presence of a permanent magnet. Table 2 lists the zeta potential data of CholPDMAEMA coated MNPs after ligand-exchange reaction, indicating that the successful surface modification of oleic acid coated MNPs. Table 2. Zeta Potential and Particle Size of Various Particles ingredients Chol-PDMAEMA coated MNPs DNA (plasmid pUC 18) Chol-PDMAEMA coated MNPs/DNAa PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNAb

zeta potential (mV) 25 −24 21 −3

± ± ± ±

2 2 1 1

Dz (nm) 118 156 110 142

± ± ± ±

6 17 2 27

a [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 20. b[CholPDMAEMA coated MNPs]/[DNA] mass ratio = 20; [MAA]/[CholPDMAEMA coated MNPs] mass ratio = 0.5.

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relatively high cytotoxicity.47,48 The aforementioned results indicated that Chol-PDMAEMA coated MNPs shows much lower cytotoxicity than branched PEI. Moreover, when PEGylated polyanion PPEGMA-b-PMAASH was added, it can reduce the cytotoxicity of polycation coated MNPs, indicating that the hydrophilic PEG chains could partially shield the positive charges of polycation and result in lower surface charge along with lower cyctoxicity. It is worth noting that at the same concentrations the HEK 293T cells viabilities are lower than those for HeLa cells. It may be ascribed to that HeLa cells are cancer cells, which have a stronger meristematic capacity and transfer capabilities, they will occupy more nutrients, and the metabolism is more active than normal HEK 293T cells. Therefore, HeLa cells exhibits higher viabilities. 3.5. Biophysical Properties of Complexes. To assess the biophysical properties of PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNA complexes, the zeta potential and size of these nanoparticles were tested, and the results are listed in Table 2. Chol-PDMAEMA with positive charges could replace the negative-charged oleic acid on MNPs by ligand-exchange reaction. Therefore, the zeta potential of these CholPDMAEMA coated MNPs showed positive charge, that was 25 ± 2 mV. Because of partial charge neutralization with negative-charged DNA, the zeta potential of Chol-PDMAEMA coated MNPs/DNA complexes ([Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 20) showed positive value about 21 ± 1 mV after combination with DNA. Subsequently, the negative-charged polyanionic brush-type copolymer PPEGMAb-PMAASH was absorbed onto the Chol-PDMAEMA coated MNPs/DNA complexes, resulting in the complexes with slightly negative zeta potentials (−3 ± 1 mV). These results prove that the PPEGMA-b-PMAASH has been successfully coated onto the CholPDMAEMA coated MNPs/DNA complexes by electrostatic interaction and shielded the positive charges as well. On the other hand, the average particle sizes of CholPDMAEMA coated MNPs in the solution was 118 nm. The naked plasmid DNA tends to form annular or linear morphology,49 which may caused that the tested size result was larger than TEM results. When Chol-PDMAEMA coated MNPs was added to interact with DNA, the average particle size of Chol-PDMAEMA coated MNPs/DNA complexes was 110 nm, demonstrating that polycation could condense DNA. Finally, PPEGMA-b-PMAASH was interacted with CholPDMAEMA coated MNPs/DNA complexes, and the size of final complexes PPEGMA-b-PMAA SH /Chol-PDMAEMA coated MNPs/DNA was about 142 nm. In order to study the colloidal stability of gene complexes, DLS was utilized to investigate the size change of complexes in the presence of salt. The results in Figure 10A showed that the sizes of gene complexes changed little in the presence of 150 mM NaCl. It can be concluded that PEGylated polyanion with cross-linkable shell could improve the colloidal stability of Chol-PDMAEMA coated MNPs/DNA complexes. Moreover, the size change of complex in the reductive environment is shown in Figure 10B. When DTT was added into the solution, the size of complex was reduced to about 90 nm. For the conventional reduction-sensitive micelles formed from disulfide linkages-containing amphiphilic copolymer, the addition of DTT will destroy the disulfide linkages, make the micelles unstable, and result in the increase of size.50 In this system, according to the schematic diagram we proposed in Scheme 1, the disulfide linkages were cleaved under the reductive

Figure 7. Effect of PPEGMA-b-PMAASH on the electrophoretic migration of DNA from Chol-PDMAEMA coated MNPs/calf thymus DNA complexes without (A) and with (B) H2O2 solution adding. Lanes 1−12 correspond to Chol-PDMAEMA coated MNPs/calf thymus DNA complexes ([Chol-PDMAEMA coated MNPs]/[DNA] = 20) with progressively increasing proportions of PPEGMA-bPMAASH ([MAA]/[Chol-PDMAEMA coated MNPs] mass ratios: 0, 0.02, 0.03, 0.05, 0.10, 0.15, 0.2, 0.3, 0.5, 0.8, 1.0, and 1.5, respectively).

with increasing concentrations of Chol-PDMAEMA MNPs, for that a high concentration of amino groups can result in

Figure 8. Gel retardation assay when DTT was added into solutions. Lanes 1−12 correspond to the in vitro degradation of CholPDMAEMA coated MNPs/calf thymus DNA complexes ([CholPDMAEMA coated MNPs]/[DNA] mass ratio = 20) with progressively increasing proportions of disulfide cross-linked PPEGMA-b-PMAASH ([MAA]/[Chol-PDMAEMA coated MNPs] mass ratios: 0, 0.02, 0.03, 0.05, 0.10, 0.15, 0.2, 0.3, 0.5, 0.8, 1.0, and 1.5, respectively). 6455


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Figure 9. Cell toxicity study by MTT assay of branched PEI (25 kDa), Chol-PDMAEMA coated MNPs and Chol-PDMAEMA coated MNPs/ polyanion complexes at various concentrations against (A) HEK 293T cells and (B) HeLa cells. [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.5.

Figure 10. Change of complex size in the presence of (A) 150 mM NaCl and (B) 10 mM DTT as a function of time, [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 20, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.5.

Figure 11. TEM images of (A) oleic acid (OA) coated Fe3O4 MNPs; (B) Chol-PDMAEMA coated MNPs; (C) DNA (plasmid pUC 18), scale bar = 100 nm; (D) Chol-PDMAEMA coated MNPs/DNA complexes at [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 20, scale bar = 500 nm; (E) PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNA complexes at [MAA]/[Chol-PDMAEMA coated MNPs] mass ratios of 0.5 and [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 20, scale bar = 200 nm.

environment and chain-exchange reaction may take place. This will weaken the electrostatic interaction between polycation

coated MNPs and DNA. Therefore, the fabricated complex was detached and the size of complex was reduced. 6456


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Figure 12. Fluorescence emission images of FITC-BSA absorbed on the surface of (A) PPEGMA-b-PMAA and (B) MePEG2000-b-PMAA observed by upright fluorescence microscope (Leica DM4000 M), excited by blue light.

Figure 13. Fluorescence microscopy images of the transfected HEK 293T cells using the PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/ DNA complexes in the serum-free medium. (A) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 3, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.05; (B) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratios = 5, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.05; (C) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 8, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.05; (D) [CholPDMAEMA coated MNPs]/[DNA] mass ratio = 10, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.05; (E) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 10, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.15; (F) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 20, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.5.

mentioned complexes with positive charges on surface, resulting in PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNA complexes, that is, the magnetic-responsive gene vector with PEGylated brush-type polymer periphery. 3.6. Anti-nonspecific Protein Adsorption Test. In order to endow the gene vector with the advantages of reducing the nonspecific adsorption with blood components, we used the brush-type copolymer as the outer layer. Here, for the purpose of testing the protein adsorption to the brush-type copolymer PPEGMA-b-PMAA (after hydrolysis from sample 5 in Table 1) in comparison with linear block copolymer MePEG2000-bPMAA obtained in our previous work,40 we utilized fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) to adsorb onto the surface of polymers films and then observed the films under the fluorescence microscopy. Figure 12A exhibits the fluorescence images of FITC-BSA absorbed on the surface of brush-type copolymer PPEGMA-b-PMAA, while Figure 12B shows that of linear copolymer MePEG2000-bPMAA. It can be seen that Figure 12A displays less green dots of FITC-BSA adsorbed to the surface of PPEGMA-b-PMAA, showing much lower protein adsorption compared with linear

TEM was further used to visualize morphology of oleic acid (OA) coated Fe3O4, Chol-PDMAEMA coated MNPs, naked DNA (plasmid pUC 18), Chol-PDMAEMA coated MNPs/ DNA complexes, and PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNA complexes as can be seen in Figure 11. Moreover, it can be observed from Figure 11E that the sizes of PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNA complexes were reduced and less than 200 nm after the addition of polyanion PPEGMA-b-PMAASH, compared with Chol-PDMAEMA coated MNPs/DNA complexes in Figure 11D. The aggregation morphology of complexes is mainly consistent with the proposed formation mechanism in Scheme 1. Chol-PDMAEMA replaced the oleic acid (OA) coated on MNPs by ligand-exchange reaction, generating the polycation coated MNPs. Chol-PDMAEMA distributed at outer layer owing to their hydrophilicity in aqueous phase. And positive charge of Chol-PDMAEMA could interact with negative charge of DNA by controlling the content of DNA to ensure the surplus positive charge on the surface of Chol-PDMAEMA coated MNPs/DNA complexes. Subsequently, the negativecharged PPEGMA-b-PMAASH was coated onto the above6457


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MePEG2000-b-PMAA in Figure 12B. This result confirms that PEGylation brush-type copolymer can reduce their nonspecific adsorption with the protein in blood. 3.7. In Vitro Transfection. The in vitro transfection efficiency was investigated in HEK 293T cells as well as HeLa cells. The fluorescence microscopy images of transfected HEK 293T cells are shown in Figure 13A−D at the mass ratios of 3, 5, 8, and 10 for [Chol-PDMAEMA coated MNPs]/ [DNA], respectively, while all the [MAA]/[Chol-PDMAEMA coated MNPs] mass ratios were 0.05. Many of green positive cells can be observed, demonstrating that these complexes have the ability to transfect into cells. Furthermore, the percentages of cells expressing enhanced green fluorescence protein (EGFP) relative to the total number of cells tested by flow cytometry measurement were 9.7%, 17.2%, 22.0%, and 33.2%, respectively. The data suggest that the cells could directly internalize the complexes and express the EGFP. It has been reported that cholesterol has a lot of advantages such as improved stability of lipid rafts and increased transfection efficiency.51,52 However, when the mass ratio of [CholPDMAEMA coated MNPs]/[DNA] increased to 20 in Figure 13F, the transfection efficiency decreased since that excessive polycation gave a lower cell viability. On the other hand, at the same mass ratio of [Chol-PDMAEMA coated MNPs]/[DNA] at 10, changing the mass ratio of [MAA]/[Chol-PDMAEMA coated MNPs] from 0.05 to 0.15, the green positive cells are reduced as shown in Figure 13E, and transgene expression level also decreased compared with Figure 13D. We attribute this phenomenon to the reason that the positive charge density of complexes reduced with increasing polyanion amount, whereas the cell surface is known to show negative and it could easily internalize the positively charged complexes. As a consequence, the complexes that possess less positive charge could not prone to get to cell surface and the endocytosis of cells was reduced, therefore resulting in the decrease of gene transfection. On the other hand, the fluorescence microscopy images of transfected HeLa cells are shown in Figure 14 at different mass ratios of [Chol-PDMAEMA coated MNPs]/[DNA] and [MAA]/ [Chol-PDMAEMA coated MNPs] without (Figure 14A−F) and with (Figure 14A′−F′) 10% serum. The results also showed the HeLa cells could directly internalize the complexes and express the enhanced green fluorescence proteins (EGFP), which are better than those for 293T cells. And the percentages of cells expressing EGFP relative to the total number of cells tested by flow cytometry measurement were 35.3%, 39.8%, 49.0%, 53.6%, 20.1%, and 50.3%, corresponding to Figure 14A−F, while were 31.1%, 36.8%, 43.0%, 27.8%, 20.5%, and 18.8% corresponding to Figure 14A′−F′, respectively. The data suggested that transfection efficiency of complex without serum was higher than that with serum. Similar to the results of 293T cells in Figure 13, the transfection efficiency and transgene expression level decreased with the increasing mass ratio of [Chol-PDMAEMA coated MNPs]/[DNA] or [MAA]/[CholPDMAEMA coated MNPs]. Furthermore, as a comparison experiment, the transfection efficiency of branched PEI (25 kDa) was investigated in HeLa cells as well in the presence of 10% serum at N/P ratio of 5, 10, 20, respectively. And the percentages of cells expressing EGFP relative to the total number of cells tested by flow cytometry measurement were 17.8%, 30.3%, and 41.2%. It can be seen that the transgene expression level of obtained complexes is close to the well-known PEI. The fluorescence microscopy images of transfected HeLa cells using PEI are shown in Figure S2

Figure 14. Fluorescence microscopy images of the transfected HeLa cells using the PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNA complexes in the serum-free culture medium (A−F) and in culture medium with 10% serum (A′−F′). (A) [CholPDMAEMA coated MNPs]/[DNA] mass ratio = 3, [MAA]/[CholPDMAEMA coated MNPs] mass ratio = 0.05; (B) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratios = 5, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.05; (C) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 8, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.05; (D) [Chol-PDMAEMA coated MNPs]/ [DNA] mass ratio = 10, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.05; (E) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 10, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.15; (F) [Chol-PDMAEMA coated MNPs]/[DNA] mass ratio = 20, [MAA]/[Chol-PDMAEMA coated MNPs] mass ratio = 0.5. Scale bar = 100 μm; the mass ratios of (A′−F′) are same with (A−F), respectively. Scale bar = 20 μm.

(Supporting Information). In general, the results indicated that the obtained stable PPEGMA-b-PMAASH/Chol-PDMAEMA coated MNPs/DNA complexes were able to transfect cells in vitro and could serve as an efficient vector in gene therapy.

4. CONCLUSIONS We have successfully synthesized a brush-type PEGylated polyanion with partial mercapto groups PPEGMA-b-PMAASH and low cytotoxic polycation Chol-PDMAEMA coated MNPs via ATRP and ligand-exchange reaction, respectively. And then a magnetic-responsive gene vector was fabricated via electrostatic interaction. Peripheral brush-type polyanion PPEGMA-bPMAASH on the vector can reduce the nonspecific adsorption with the protein in blood. Moreover, the disulfide linkages (S− S) between the copolymer PPEGMA-b-PMAASH were formed by oxidizing the mercapto groups to stabilize the DNA complexes, and it could be cleaved and release DNA in a cell reducing environment. The results of zeta potential, DNA binding capacity, antinonspecific protein adsorption test, cytotoxicity assay, DLS and TEM measurements, and in vitro transfection tests unveil that this gene vector showed magnetic6458


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responsive, anti-nonspecific protein adsorption, low cytotoxicity, and could realize transfection on HEK 293T and HeLa cells. Consequently, it has potential applications for the targeted intracellular delivery in gene therapy.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of PPEGMA homopolymer before and after TFA treatment; fluorescence microscopy images of transfected HeLa cells using PEI. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (20974074 and 21074078), Natural Science Foundation of Jiangsu Province for Rolling Support Project (BK2011045), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Province Key Laboratory of Stem Cell Research, Soochow University. Ying Hao and Jinlin He have respectively received the financial support from the Innovation Project of Graduate Students of Jiangsu Province, China (CXZZ11_0091 and CX09B_021Z). We are also thankful to Dr. Zhuang Liu (FUNSOM, Soochow University), Prof. Mingde Qin, and Miss Hansi Liang (Medical College of Soochow University) for their valuable help in in vitro cytotoxicity assay and in vitro transfection tests.

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REFERENCES

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