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Well Defined Peapod-like Magnetic Nanoparticles and Their Controlled Modification for Effective Imaging Guided Gene Therapy Ranran Wang, Yang Hu, Nana Zhao, and Fujian Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01697 • Publication Date (Web): 21 Apr 2016 Downloaded from http://pubs.acs.org on April 24, 2016

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ACS Applied Materials & Interfaces

Well Defined Peapod-like Magnetic Nanoparticles and Their Controlled Modification for Effective Imaging Guided Gene Therapy Ranran Wang,a,b,c Yang Hu,a,b,c Nana Zhao,*a,b,c and Fu-Jian Xu*a,b,c a

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China b Key Laboratory of Carbon Fiber and Functional Polymers (Beijing University of Chemical Technology), Ministry of Education, Beijing, 100029, China c Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing, 100029, China * To whom correspondence should be addressed E-mail addresses: [email protected] (Nana Zhao), [email protected] (Fu-Jian Xu).

KEYWORDS: peapod-like, magnetic nanoparticles, polycation, gene transfection, MRI imaging.

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ABSTRACT: Due to their unique properties, one-dimensional (1D) magnetic nanostructures

are of great significance for bio-related applications. A facile and straightforward strategy to fabricate 1D magnetic structure with special shapes is highly desirable. In this work, well defined peapod-like 1D magnetic nanoparticles (Fe3O4@SiO2, p-FS) are readily synthesized by a facile method without assistance of any templates, magnetic string or magnetic field. There are few reports on 1D gene carriers based on Fe3O4 nanoparticles. BUCT-PGEA (ethanolamine-functionalized poly(glycidyl methacrylate) is subsequently grafted from the surface of p-FS nanoparticles by atom transfer radical polymerization to construct highly efficient gene vectors (p-FS-PGEA) for effective biomedical applications. Peapod-like p-FS nanoparticles were proven to largely improve gene transfection performance compared with ordinary spherical Fe3O4@SiO2 nanoparticles (s-FS). External magnetic field was also utilized to further enhance the transfection efficiency. Moreover, the as-prepared p-FS-PGEA gene carriers could combine the magnetic characteristics of p-FS to well achieve noninvasive magnetic resonance imaging (MRI). We show here novel and multifunctional magnetic nanostructures fabricated for biomedical applications that realized efficient gene delivery and real-time imaging at the same time.

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INTRODUCTION Fe3O4 nanoparticles with the unique features of controllable sizes, fast magnetic response, low cytotoxicity and biocompatibility, have attracted considerable attention in diverse bio-related applications, including bio-separation, magnetic guided delivery, gene carriers, and magnetic resonance imaging (MRI).1-3 Therefore, Fe3O4 nanoparticles are ideal candidates for theranostics combining the dual functions of therapy

and

imaging

for

disease

diagnosis

and

treatment

monitoring.4-6

One-dimensional (1D) nanostructures are of great significance because of their favorable properties along with potential applications.7 Moreover, they offer the simplified models to exploit the influence of size and shape of nanoparticles on different aspects of properties and applications. Many efforts had been devoted to the synthesis of 1D magnetic nanoparticles, especially through assembly with the assistant of magnetic field, where a magnetic field is utilized to induce the nanoparticles to 1D structures due to strong magnetic dipolar interactions.8 Although this method seems powerful, how to retain the assembled structures still remains great challenge when the magnetic field is moved away. To obtain permanent 1D structure, a variety of inorganic nanoparticles and polymers had been utilized to coat and stabilize the magnetic chains.9-12 Notably, well-defined magnetite nanoparticles were realized by connecting the assembled chains of silica-coated magnetic nanoparticles with titanium oxide.13 However, such process underwent multiple steps with the assistance of magnetic field. A facile and straightforward avenue to fabricate 1D structure with permanent connection between nanoparticles would be desirable. Multifunctional gene carriers based on Fe3O4 nanoparticles hold great promises for the noninvasive diagnosis and therapy of cancer since MRI detection will be applied within the same nanostructure.14-19 The transfection performance is assumed to be

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enhanced significantly by magnetofection based on superparamagnetic nanoparticles, where the accumulation and cellular internalization of magnetic carriers/gene complexes could be facilitated by external magnetic fields.20,21 The size, shape and surface functionalization of nanoparticles were demonstrated to have significant influence on gene transfection performance, probably by affecting cellular uptake.22,23 It was reported that 1D nanoparticles with higher aspect ratios than ordinary spherical counterparts do favor cellular uptake.24,25 In this regard, we attempt to modulate these factors of Fe3O4 nanoparticles to achieve satisfying gene carriers. There are few reports on 1D gene carriers based on Fe3O4 nanoparticles while spherical nanostructures were mostly employed. Lately, we exploited a kind of efficient gene carriers with compromised cytotoxicity based on BUCT-PGEA.26,27 These results inspire us to fabricate highly efficient gene carriers based on 1D Fe3O4 structures functionalized with BUCT-PGEA for theranositics. In the present study, novel 1D peapod-like magnetic nanoparticles (Fe3O4@SiO2, p-FS) were readily synthesized using a highly reproducible one-step method to coat Fe3O4 nanoparticles with silica in the absence of external fields. The synthesis strategy developed is mild and straightforward with products with high yields and does not involve any templates or magnetic fields. In addition, the outer silica layer could promote the chemical stability of the magnetic nanoparticles. Spherical Fe3O4@SiO2 magnetic nanoparticles (s-FS) were prepared as the control counterparts. BUCT-PGEA was then grafted from the surface of p-FS via ATRP to fabricate p-FS-PGEA organic/inorganic hybrids (Figure 1). Then, the detailed gene transfection and MRI were investigated in vitro and in vivo to establish therapeutic and diagnostic applications of the as-prepared p-FS-PGEA hybrids. Magnetofection was manipulated in the system to further enhance the performance. A promising platform combining

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gene therapy and real-time imaging has been proposed via the integration of the MRI function of 1D peapod-like magnetic nanoparticles.

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EXPERIMENTAL SECTION Materials. Sodium citrate (NaCit, 99%), ethanol (99%), sodium acetate (NaAc, 99%), ethylene glycol (EG, 96%), dichloromethane (CH2Cl2, 99.5%), and aqueous ammonia (NH4OH, 25wt %, 99%) were purchased from Beijing Chemical Co., China. Ferric chloride (FeCl3•6H2O, 99%) was obtained from Beijing Modern Oriental Fine Chemistry

Co.,

Ltd,

China.

3-aminopropyl

triethoxysilane

(APTES)

and

tetraethylorthosilicate (TEOS, 98%) were obtained from Energy Chemical Co., Ltd, Shanghai, China. Copper (I) bromide (CuBr, 98%), branched polyethylenimine (PEI, Mw ∼25KDa), glycidyl methacrylate (GMA, 98%), 2-bromoisobutyryl bromide (BIBB, 98%), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), triethylamine (TEA), ethanolamine (EA, 98%), penicillin, N,N,N',N,'N''-pentamethyl diethylenetriamine, and streptomycin were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO. HepG2 and C6 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). The plasmid (encoding Renilla luciferase, pRL-CMV) was from Promega Co., while Cergy Pontoise and plasmid pEGFP-N1 (encoding enhanced green fluorescent protein (EGFP)) were from BD Biosciences, San Jose, CA. Synthesis of FS Nanoparticles. Fe3O4 nanoparticles were prepared using a typical solvothermal method.28 For the synthesis, NaCit acts as surfactant and FeCl3 was reduced by EG at 200 oC. 0.325 g of FeCl3•6H2O was dissolved in 20 mL of EG, and 0.2 g of NaCit was added with stirring. Then, 1.2 g of NaAc was added with continuous stirring for 1 h. Thereafter, the mixed solution was added into stainless-steel autoclave before heated at 200 oC. The products were collected by magnet separation and washed with ethanol and water. With the reaction of 8 h and 10 h, Fe3O4 nanoparticles of ~80 nm and ~200 nm in diameter were achieved. The p-FS 6


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nanoparticles were synthesized as follows. 1 mL of Fe3O4 (∼80 nm) solution was dissolved in 28 mL of ethanol and 6 mL of water. Then, 2.5 mL of ammonia were introduced. Afterwards, three 50 µL injection of TEOS was added with continuous stirring at 30 min intervals and the reaction last for 10 h to produce p-FS nanoparticles. The synthesis of s-FS nanoparticles from ~200 nm Fe3O4 followed similar procedure except that the amount of silica source was adjusted to three 40 µL injection of TEOS. Immobilization of ATRP Initiator on FS Surfaces. FS was first modified by APTES to form FS-NH2 with the terminal amino groups.29 Then, FS-NH2 was reacted with BIBB to produce FS-Br. 0.1 g of FS was added in the mixed solvents of 9 mL of ethanol and 1mL of water. 0.2 mL of APTES was dissolved into the mixed solution with stirring for 6 h. Then, 0.2 mL of TEA was added and stirred for another 18 h. The precipitate was separated by centrifugation before washing with water and ethanol for three times. FS-Br was synthesized by reacting FS-NH2 with BIBB. 0.1 g of FS-NH2 was dissolved in 10 mL of CH2Cl2, and then 1.5 mL of BIBB together with 1.5 mL of TEA were added. After 3 h, centrifugation at 10000 rpm was employed to purify the products. The final product was freeze-dried. Preparation of FS-PGEA Nanoparticles. Firstly, FS-PGMA nanoparticles were produced via ATRP of GMA employing FS-Br as the initiator. For the typical synthesis of p-FS-PGEA1, 20 mg of FS-Br was added in 4 mL of DMSO. Then 0.5 mL of GMA was dissolved in the mixture, followed by the addition of 32 µL of PMDETA. 7 mg of CuBr was introduced into the mixture and the reaction was maintained for 20 min with continuous stirring. The final reaction was terminated by opening the system to air. The products were collected by precipitation. The preparation of EA-functionalized FS-PGMA (FS-PGEA) was described as follows. FS-PGMA was dissolved in the mixture of 2 mL of DMSO and 0.5 mL of EA. After 7



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the mixture was degassed with N2 for 10 min, the reaction was maintained for 24 h, and then transferred to oil bath at 80 oC for 40 min. Nanohybrids of p-FS-PGEA2, s-FS-PGEA1 and s-FS-PGEA2 were synthesized employing similar procedure except

that the amount of GMA and ATRP reaction time were fixed as 1 mL of GMA for 40 min, 0.5 mL of GMA for 25 min, and 1 mL of GMA for 50 min ATRP, respectively. The product was purified by dialysis. Characterization. The nanohybrids of FS-PGEA were characterized by transmission electron microscopy (TEM, FEI Company, Hillsboro, OR, 200 KV), X-ray photoelectron spectroscopy (XPS, Kratos AXIS HSi, Al Kα), thermal gravimetric analysis (TGA, TGA/DSC 1/1100 SF, Mettler Toledo, Switzerland), atomic force microscopy (AFM, Bruker Dimension Icon, Bruker, Santa Barbara, CA)， as well as particle size and zeta potential measurements (Zetasizer Nano ZS, Malvern Instruments, Southborough, MA). Agarose gel electrophoresis (Bio-Rad Lab, Hercules, CA) was used to test the ability of FS-PGEA nanohybrids to bind pDNA.30 FS-PGEA stock solutions were stored at 4 oC with the nitrogen concentration of 10 mM. Desirable N/P ratio (from 0 to 3.5) was obtained by mixing various amounts of FS-PGEA stock solutions with 0.2 µg of pDNA solution. As a result, the concentration of FS-PGEA1 increased from 0 to 53 µg/mL while FS-PGEA2 from 0 to 30 µg/mL. The images of DNA bands were captured by a UV transilluminator and BioDco-It imaging system (UVP Inc, Upland, CA). Cytotoxicity Assay. The cytotoxicity of FS-PGEA/pDNA was tested by MTT viability assay in C6, HepG2 and HEK293 cell lines. As previously reported,31,32 serial dilutions of FS-PGEA/pDNA nanocomplexes at N/P ratios from 5 to 30 were prepared by mixing 0.3 µg of pDNA with various amounts of FS-PGEA. The concentration of FS-PGEA1 per well increased from 25 to 150 µg/mL, while 8


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FS-PGEA2 from 14 to 87 µg/mL, respectively. Transfection Assay. Transfection assays were carried out in C6 and HepG2 cell lines utilizing plasmid pRL-CMV as the reporter gene. The procedure is similar to our previous reports.23,32 The FS-PGEA/pDNA complexes at various N/P ratios were prepared by mixing FS-PGEA with 1.0 µg of pDNA. The concentration of FS-PGEA1 per well increased from 19 to 108 µg/mL while FS-PGEA2 from 11 to 62 µg/mL, respectively. The transferred cells were lysed with lysis reagent (Promega Co., Cergy Pontoise, France). For transfection in the presence of magnet, the culture plates were placed on the applied magnets for 4 h after nanocomplexes were introduced into the transfection medium. In order to get intuitive gene expression, plasmid pEGFP-N1 was employed as another reporter gene following the procedure in earlier report.23, 32 Determination of Cellular Internalization. The procedure of determination of cellular internalization was similar to our previous work33 and HepG2 cells were used as the representative. The complex containing 78 µg of FS-PGEA2 and 6 µg of labeled pDNA (N/P ratio of 15) was added to each well. For the group applied with magnets, the plates were placed on the magnets and incubated for 4 h. The test procedure was similar to our previous work.34 MRI Assay. For in vitro assay, approximately 5×106 HepG2 cells were seeded in a 5 mL cell culture flask. The cells were then incubated with p-FS-PGEA2 at various Fe concentrations (0, 7.5, 15, and 30 µM, respectively) for 4 h with or without applying the magnet. Then, the cells were washed, trypsinized, centrifuged, and resuspended in 0.2 mL of PBS in Eppendorf tubes. The MRI experiments were performed on a 7.0-T MRI instrument (BioSpec 70/20 USR 7.0 T Bruker). The following parameters were adopted: repetition time (TR) of 3000 ms; echo time (TE) of 50 ms; field of view (FOV) read of 3.5 cm2; matrix: 256 × 256; number of excitations (NEX): 2; slice 9



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thickness = 1 mm. For in vivo MRI measurements, 100 µL of p-FS-PGEA2/pDNA complexes with the Fe concentration of 20 mM at the N/P ratio of 15 was intravenously injected via tail vein. MR images of liver and kidney were collected at different time intervals (0, 30 and 60 min) after intravenous injection. Parameters adopted: TR/TE = 3000/50 ms; FOV: 4 cm2; matrix: 256×256; slice thickness = 1 mm.

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RESULTS AND DISCUSSION Fabrication of Peapod-like Magnetic Nanoparticles. The design rationale of peapod-like Fe3O4@SiO2 magnetic nanoparticles (p-FS) is to encapsulate two or more Fe3O4 nanoparticles into 1D structure with a layer of silica. A typical solvothermal method was first employed to synthesize Fe3O4 nanoparticles.28 The as-prepared Fe3O4 nanoparticles were well dispersed and their sizes could be adjusted from 80 to 410 nm by changing reaction time.28 Since the cellular uptake of iron oxide would be limited for particles larger than 300 nm,35 Fe3O4 nanoparticles of 80 nm were utilized to fabricate peapod-like structure. As shown in Figure 2, two kinds of Fe3O4 nanoparticles with different sizes could be obtained: ∼80 nm (Figure 2a), and ∼200 nm in diameter (Figure 2d). Fe3O4 nanoparticles of ∼80 nm were employed to prepare p-FS through classical Stöber method36. As shown in Figure 2b and c most p-FS nanoparticles incorporate two Fe3O4 nanoparticles in a line to manifest the peapod-like structure. The diameter of the p-FS was ∼160 nm while the length ~280 nm. Ordinary spherical magnetic nanoparticles (s-FS) were also synthesized using Fe3O4 nanoparticles of ∼200 nm, where a single Fe3O4 nanoparticle was encapsulated in silica. In both cases, the thickness of silica wall was fixed to be around 40 nm and the diameter of s-FS was ~280 nm (Figure 2e and f), comparable to the length of p-FS. Since it has been reported that 1D nanoparticles facilitate cellular uptake and gene delivery compared with spherical ones possessing identical diameter,23,37 s-FS with the diameter comparable to the diameter of p-FS was not investigated further. For this strategy, the number of Fe3O4 cores encapsulated in silica was controlled by the number concentration of Fe3O4 nanoparticles. It is supposed that the movability of 80 nm Fe3O4 nanoparticles was relatively higher than that of 200 nm nanoparticles. In this case, two or more particles tend to stay in one structure probably resulting from 11



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the increase in collisions among particles during the hydrolysis and condensation processes of TEOS. Finally, 1D peapod-like p-FS nanoparticles were achieved for the following surface functionalization and biomedical applications while spherical s-FS counterparts with the similar overall size were synthesized for comparison. Synthesis and Characterization of FS-PGEA Nanohybrids. The FS-PGEA nanohybrids of the two types of FS were synthesized by introducing BUCT-PGEA gene carriers on the nanoparticle surfaces via ATRP (Figure 1). The immobilization of ATRP initiator is crucial for the functionalization of FS. The surface of FS was firstly modified by 3-aminopropyl-triethoxysilane (APTES),38 producing FS-NH2. Then, the -NH2 groups of FS-NH2 reacted with BIBB, resulting in the bromoisobutyryl group-terminated FS (FS-Br) which could act as ATRP initiator. FS-PGEA nanohybrids were then fabricated via ATRP of glycidyl methacrylate (GMA) from FS-Br (Figure 1). Finally, FS-PGEA could be obtained through ring-opening reaction with excess ethanolamine (EA). Through varying the amount of GMA and ATRP time, the length of grafted BUCT-PGEA chains could be controlled. Here, we fabricated two kinds of FS-PGEA with different weight ratios of BUCT-PGEA. XPS was utilized to verify the functionalization processes on FS nanoparticles (Figure 3). Taking p-FS nanoparticles for example, the signals from Si and O of SiO2 dominated the elemental peaks before modification (Figure 3a). The formation of FS-NH2 was confirmed by the appearance of N 1s signal (at the binding energy (BE) of 399 eV, attributable to the amine (-NH2) species39) (Figure 3b and b’). As a characteristic of covalently bonded bromine, Br 3d signal (BE of 69 eV) indicated that the alkyl halide group was introduced successfully (Figure 3c and c’). When PGMA was grafted on the surface, the signals of Si decreased substantially, which was caused by the thick PGMA layer on the surface compared with the former FS-NH2 and FS-Br.

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At the same time, the signal of C was much stronger, as shown in Figure 3d. After the reaction with EA, much stronger N signal was observed, confirming the formation of FS-PGEA (Figure 3e). Thermal gravimetric analysis (TGA) was carried out to determine the weight ratio of immobilized FS-Br and BUCT-PGEA of FS-PGEA. The TGA curves (Figure S1) demonstrate that there was ~10% mass loss for the pristine FS nanoparticles, probably resulting from the dehydration and condensation of -OH of SiO2 shell. The TGA curves of FS-Br suggest 82 wt% FS solid was left after the removal of organics at around 600 oC. The amounts of BUCT-PGEA in the two kinds of FS-PGEA were calculated to be around 40 wt% and 70 wt%, namely FS-PGEA1 and FS-PGEA2, respectively. DNA Condensation Capability of FS-PGEA Nanohybrids. It is essential for FS-PGEA nanohybrids to condense pDNA and form compact FS-PGEA/pDNA complexes for efficient gene delivery. In the present work, we evaluate DNA condensation capabilities of FS-PGEA nanohybrids by agarose gel electrophoresis, AFM imaging, together with particle size and zeta potential measurements. N/P ratio was used to present the molar ratio of FS-PGEA to DNA.23 The electrophoretic mobility of FS-PGEA/pDNA complexes was first tested at N/P ratios from 0 to 3.5 (the concentration of FS-PGEA1 increased from 0 to 53 µg/mL while FS-PGEA2 from 0 to 30 µg/mL). After FS was functionalized with cationic BUCT-PGEA, the FS-PGEA nanohybrids with positive charges could condense negatively charged pDNA partially or completely, which caused the retardation of the pDNA.40 The gel retardation result of FS-PGEA/pDNA nanocomplexes is shown in Figure 4a. It could be observed that all FS-PGEA nanohybrids of two different morphologies with 40 wt% or 70 wt% BUCT-PGEA could compact pDNA effectively within the N/P ratio of 2

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and demonstrated similar DNA-condensing capability. The above results verified successful surface functionalization of FS with BUCT-PGEA and their excellent capability for DNA condensation. In addition, the hydrodynamic particle sizes and surface potentials of complexes were measured by dynamic light scattering measurements, which play a significant role in cytotoxicity, cellular uptake, and the release of pDNA.41-44 Figures 4b,c show the hydrodynamic particle sizes and surface potentials of the FS-PGEA/pDNA complexes. The pristine p-FS-PGEA1 and s-FS-PGEA1 demonstrated the sizes around 500 nm. When the amount of BUCT-PGEA in FS-PGEA nanohybrids increased, the sizes of the nanohybrids (p-FS-PGEA2 and s-FS-PGEA2) increased to be around 600 nm (Figure 4b). After mixing various amounts of pristine FS-PGEA with pDNA for 30 min, FS-PGEA/pDNA complexes with different N/P ratios were formed. As shown in Figure 4b, all the FS-PGEA nanohybrids could compact pDNA into smaller particles compared with pristine FS-PGEA due to the shrinkage of BUCT-PGEA chains. With the content of FS-PGEA (or N/P ratio) increased, the size of the complexes tended to increase. With increasing the N/P ratio, excess cationic FS-PGEA caused the increase in size. The surface of FS-PGEA/pDNA complexes is positively charged which would facilitate cellular uptake through good affinity with cell membranes which is negatively charged. As displayed in Figure 4c, the zeta potential of all FS-PGEA/pDNA complexes remained positive and increased slightly with the N/P ratio, which would facilitate cell internalization.45 The size and shape of FS, FS-PGEA and FS-PGEA/pDNA were further characterized by AFM imaging. After the surface of FS was functionalized with 70 wt% of BUCT-PGEA, p-FS-PGEA2 and s-FS-PGEA2, together with p-FS-PGEA2/pDNA and s-FS-PGEA2/pDNA were imaged by AFM (Figure 5). The AFM image of p-FS

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clearly revealed the peapod-like morphology (Figure 5a). After functionalization, the morphology of p-FS-PGEA2 remained the 1D characteristic while the diameter of particles increased substantially (Figure 5a and b). After pDNA was complexed, the diameter of p-FS-PGEA2/pDNA at the N/P ratio of 20 decreased slightly (Figure 5c). This result indicated that BUCT-PGEA brushes compacted pDNA, in agreement with the change of particle sizes (Figure 4b). For s-FS, similar phenomenon was observed that s-FS-PGEA2 and s-FS-PGEA2/pDNA complexes retained spherical shapes while the particle sizes demonstrated the same propensity to increase firstly and decrease subsequently (Figure 5d-f). The AFM results confirm that FS-PGEA2 could compact pDNA effectively while preserving the morphologies of FS. Cell Viability Assay of FS-PGEA. It is necessary to evaluate the cytotoxicity of the FS-PGEA carriers before utilized for gene delivery. The cell viability of the FS-PGEA/pDNA complexes was assessed by MTT assay. In this work, the difficult-to-transfect HepG2 and C6 cells, as well as normal health HEK293 cells were selected as the model cell lines.46 With increasing the concentration of FS-PGEA nanohybrids, the cell viability of all complexes fabricated from s-FS and p-FS with 40 wt% and 70 wt% BUCT-PGEA gradually decreased, as shown in Figures 6a and S2. When increasing the N/P ratio from 5 to 30, the amount of FS-PGEA1 increased from 25 to 150 µg/mL while FS-PGEA2 increased from 14 to 87 µg/mL. The free cationic FS-PGEA nanohybrids at higher N/P ratios probably increased cell cytotoxicity. Complexes of s-FS-PGEA/pDNA and p-FS-PGEA/pDNA with the same amount of BUCT-PGEA demonstrated similar cytotoxicity when the N/P ratio was identical. The cytotoxicity of gene carriers was observed to depend on the weight ratio of BUCT-PGEA for both s-FS and p-FS. Generally speaking, FS-PGEA2 with 70 wt% BUCT-PGEA was slightly more toxic than FS-PGEA1 with 40 wt% BUCT-PGEA,

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consistent with the fact that the cytotoxicity of polycations increase with the molecular weight.31 It is to be noted that in both cell lines all the FS-PGEA nanohybrids displayed remarkably lower cytotoxicity than the control “gold standard” PEI (Mw~25KDa)23,32 at various N/P ratios. In Vitro Gene Transfection Assay Mediated by FS-PGEA. Luciferase was first employed as the reporter gene to investigate the gene transfection efficiency of FS-PGEA/pDNA complexes in both HepG2 and C6 cell lines. The gene transfection efficiencies were found to be influenced by several factors, such as N/P ratio, morphology, weight ratio of BUCT-PGEA and the presence of external magnetic field, as shown in Figure 6b. When the N/P ratio increased from 5 to 30, the concentration of FS-PGEA1 increased from 19 to 108 µg/mL while FS-PGEA2 increased from 11 to 62 µg/mL. As the N/P ratio was above 15, transfection efficiencies mediated by most FS-PGEA nanohybrids were higher compared with PEI at its optimal N/P ratio of 10,23,32 indicating their promising potential for gene delivery. In HepG2 cells, the optimal gene transfection efficiency of all FS-PGEA nanohybrids was at the N/P ratio of 20 while in C6 cells it appeared at the N/P ratio of 15. For lower N/P ratios, pDNA could not be condensed efficiently by FS-PGEA nanohybrids due to relatively low amount of positive charges. For higher N/P ratios, the presence of excess FS-PGEA nanohybrids would induce cytotoxicity, probably resulting in modest decrease of transfection efficiency (Figure 6a). Moreover, the transfection efficiencies mediated by FS-PGEA2 were higher than those mediated by FS-PGEA1 at the same N/P ratio, indicating that the transfection efficiencies of FS-PGEA nanohybrids was significantly influenced by the length of BUCT-PGEA. This result is consistent with the general knowledge that polycations with high molecular weight demonstrate higher gene transfection efficiency.23,32

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It is worth mentioning that p-FS-PGEA as gene carriers exhibited obviously superior quality than s-FS-PGEA, implying that gene transfection performance was influenced by the morphology of nanohybrids. This observation also verified our hypothesis that 1D p-FS with larger aspect ratio than s-FS does favor transfection performance. In addition, 2-3 times higher transfection efficiency than those of normal culture conditions was achieved for FS-PGEA2 with both morphologies when a magnetic field was applied (p-FS-PGEA2 (M) and s-FS-PGEA2 (M)). At the identical N/P ratio, p-FS-PGEA2 nanohybrids under magnetic field demonstrated best transfection performance, especially at lower N/P ratios. In order to further compare the gene delivery of FS-PGEA carriers, enhanced green fluorescent protein (EGFP) was performed for direct visualization of gene expression. Figure 7 shows the representative fluorescence images of EGFP gene expression in HepG2 cells mediated by p-FS-PGEA2 as well as s-FS-PGEA2 with or without magnetic field at the N/P ratio of 20. The transfection efficiency could be reflected by percentage of green EGFP-positive cells and was obtained using flow cytometry. In the absence of magnetic field, p-FS-PGEA and s-FS-PGEA2 represented the percentage of 38% and 30%, respectively. With magnetic field, p-FS-PGEA2 (M) showed the highest percentage of 45% while 40% for s-FS-PGEA2 (M). These findings again confirmed the superiority of p-FS-PGEA carriers, in agreement with the luciferase gene transfection (Figure 6b). Cellular Internalization. The cellular internalization of FS-PGEA2/pDNA complexes in HepG2 cell line with and without magnetic field was investigated to verify the speculation that morphology and magnetic field influences the internalization of magnetic carriers. YOYO-1 was used to label pDNA in green and DAPI was employed to stain nuclei in blue. Figure 8a shows that the HepG2 cells

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treated with p-FS-PGEA2/pDNA presented stronger green pDNA signals than s-FS-PGEA/pDNA. The greater internalization amounts may result from larger contact area of 1D p-FS-PGEA2/pDNA with the cell membrane than the spherical s-FS-PGEA/pDNA. After magnetic field was applied, the signals from p-FS-PGEA2 (M)/pDNA and s-FS-PGEA2 (M)/pDNA complexes were enhanced further compared with their counterparts with free magnetic field. The rate of cellular internalization was obtained by flow cytometry. As shown in Figure 8b, the p-FS-PGEA (M)/pDNA (or s-FS-PGEA (M)/pDNA) complexes displayed the rates of 95% (or 89%), while the p-FS-PGEA/pDNA (or s-FS-PGEA/pDNA) complexes presented relatively lower rates of 84% (or 75%). These results reveal that both peapod-like morphology and magnetic field could facilitate cellular uptake. The 1D and magnetic virtues of p-FS-PGEA carriers might favor their internalization and endow them with superior gene transfection performance. MRI Assay. The magnetic feature of FS-PGEA carriers renders them attractive MRI contrast agent. Due to its better gene transfection performance, p-FS-PGEA2 was selected as the typical example for the following MRI assay. Here, aqueous solutions of p-FS-PGEA2 nanohybrids with various concentrations of Fe were first examined by T2-weighted MRI. As displayed in Figure 9a, the intensity of T2 signal decreased obviously with Fe concentration resulting from the dipolar interaction of the protons in water with magnetic moments of p-FS-PGEA2.47 The transverse relaxivity (r2) value of p-FS-PGEA2 was estimated to be 29.88 mM-1s-1 (Figure 9a’). The relatively low r2 value was probably caused by the coating layers of SiO2 and BUCT-PGEA on the surface of Fe3O4 nanoparticles.48,49 Then, T2-weighted MR images of HepG2 cells mediated with p-FS-PGEA2 were also characterized and found to become darker and darker with increasing Fe concentration (Figure 9b).

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When external magnetic field was applied, the MR images of HepG2 cells also followed similar manner, where the dark intensity was dependent on Fe concentration. Meanwhile, significantly reduced intensity of signals was observed, which is in agreement with the results of cellular internalization. That is to say, the external magnetic field could not only facilitate gene transfection but also improve the spatial resolution for MRI applications. The excellent MR imaging capability of the p-FS-PGEA2 carriers in vitro encouraged us to further explore the in vivo contrast enhancement effect. Figure 10a show the MR images of nude mice before as well as 30 min and 60 min after intravenous injection of p-FS-PGEA2/pDNA. Generally speaking, the carriers tend to mainly accumulate in the liver tissue. In this work, it is obvious that the liver and kidney got darker with time after intravenous injection. After 60 min, efficient dark contrast in T2 imaging of the liver and kidney could be distinguished, suggesting p-FS-PGEA2 could be employed as T2 contrast agent for MR imaging of mouse liver and kidney in vivo. Figure 10b provides the value of 1/T2 with the injection time, confirming the above changes in signal intensities. The corresponding in vivo gene transfection in different organs was also assayed (Figure S3). The gene expression levels mediated by p-FS-PGEA2 were higher in liver and kidney than other organs, which verifies the MR imaging results in vivo. Both in vitro and in vivo data indicate p-FS-PGEA nanohybrids were promising gene carriers and contrast agents for MRI, which could be utilized for early detection and response tracking of gene therapy.

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CONCLUSIONS In this work, novel 1D peapod-like p-FS magnetic nanoparticles were successfully synthesized using a facile strategy without involving any templates or magnetic fields. The controllable surface functionalization of p-FS with BUCT-PGEA was realized via surface-initiated ATRP to fabricate the multifunctional gene carrier (p-FS-PGEA) integrating gene delivery and MR imaging. Compared with ordinary spherical counterparts (s-FS-PGEA), p-FS-PGEA carriers demonstrated superior gene transfection performances in both HepG2 and C6 cells. Such 1D peapod-like shape could facilitate cellular uptake. Under magnetic field, the transfection efficiency could be further enhanced. In addition, p-FS-PGEA could effectively combine the magnetic characteristics of p-FS and exhibited excellent performance in MR imaging in vitro and in vivo. The present work offers a versatile approach to utilize 1D magnetic nanoparticles for the construction of multifunctional carriers.

ASSOCIATED CONTENT Supporting Information Thermogravimetric analysis and in vivo gene transfection data of p-FS-PGEA-based nanostructures can be found in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author E-mail addresses: [email protected], [email protected]. 20


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Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENTS This work was supported by National Natural Science Foundation of China (grant numbers 51173014, 51221002, 51373017, 51325304, 51302009 and 51473014), BUCT Fund for Disciplines Construction and Development (Project No. XK1513), Innovation and Promotion Project of Beijing University of Chemical Technology, and Collaborative Innovation Center for Cardiovascular Disorders, Beijing Anzhen Hospital Affiliated to the Capital Medical University.

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Figures

Figure 1. Schematic illustration of the fabrication processes of PGEA-grafted Fe3O4@SiO2 nanohybrids (FS-PGEA) via ATRP.

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Figure 2. TEM images of the Fe3O4 nanoparticles (a,d), p-FS (b,c), and s-FS (e,f).

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Figure 3. Typical XPS wide-scan spectra of (a) p-FS, (b) p-FS-NH2, (c) p-FS-Br, (d) p-FS-PGMA, and (e) p-FS-PGEA together with (b’) N 1s core-level spectrum of p-FS-NH2 and (c’) Br 3d core-level spectrum of p-FS-Br.

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Figure 4. Electrophoretic mobility retardation assay of pDNA (a) in the complexes with (a1) p-FS-PGEA1, (a2) p-FS-PGEA2, (a3) s-FS-PGEA1, and (a4) s-FS-PGEA2 at different N/P ratios as well as particle sizes (b) and zeta potentials (c) of the FS-PGEA/pDNA complexes at different N/P ratios compared with s-FS and p-FS. (mean ± SD, n=3)

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Figure 5. AFM images of (a) p-FS, (b) p-FS-PGEA2, (c) p-FS-PGEA2/pDNA (N/P = 20), (d) s-FS, (e) s-FS-PGEA2, and (f ) s-FS-PGEA2/pDNA (N/P =20).

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Figure 6. MTT viability assay of HepG2 (a1) and C6 (a2) cell lines mediated with FS-PGEA/pDNA and PEI/pDNA at various N/P ratios. Luciferase gene transfection efficiency of the FS-PGEA/pDNA complexes at different N/P ratios compared with PEI in HepG2 (b1) and C6 (b2) cell lines.

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Figure 7. Microscopic images of EGFP gene transfection treated by p-FS-PGEA2 (a,a’), s-FS-PGEA2 (b,b’), p-FS-PGEA2 (M) (c,c’), and s-FS-PGEA2 (M) (d,d’) at the N/P ratio of 20 in HepG2 cell line. Scale bar: 100 µm.

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Figure 8. Fluorescence images (a) and flow cytometry analysis (b) of HepG2 cells treated with s-FS-PGEA/pDNA, p-FS-PGEA/pDNA, s-FS-PGEA (M)/pDNA, and p-FS-PGEA (M)/pDNA at the N/P ratio of 20 for 4 h. Scale bar: 50 µm.

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Figure 9. (a) MR images and (a’) linear fitting of 1/T2 of p-FS-PGEA2 in aqueous solutions with different concentrations of Fe, as well as (b) MR images and (b’) corresponding 1/T2 of HepG2 cells treated with p-FS-PGEA2 for 4 h at different Fe concentrations.

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Figure 10. (a) MR images and (b) 1/T2 of liver and kidney of nude mice before as well as 30 min and 60 min after intravenous injection of p-FS-PGEA2/pDNA.

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