SPIO Nanocomplexes ... - ACS Publications

May 19, 2015 - The pDNA were amplified in DH5-α competent cell and purified by NucleoBond. Xtra Midi EF Kit (Macherey Nagel, Düren, Germany). Dulbec...
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Redox-Sensitive Polymer/SPIO Nanocomplexes for Efficient Magnetofection and MR Imaging of Human Cancer Cells Rih-Yang Huang, Pin-Hsin Chiang, Wei-Chen Hsiao, Chun-Chiao Chuang, and Chien-Wen Chang* Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, 30013, Taiwan S Supporting Information *

ABSTRACT: Magnetofection has received increasing attention for its great potential on gene therapy. To promote its clinical therapeutic applications, development of safe and effective magnetic nanocarriers is in high demand. Herein, we present a redox-sensitive polymer/metal nanocomplex system (PSPIO) for efficient magnetofection and magnet resonance imaging (MRI) on cancer cells. PSPIO was prepared by modifying SPIO with redox-sensitive polyethylenimine (SSPEI) via a ligand exchange process. PSPIO could efficiently condense plasmid DNA (pDNA) into nanoparticles, which exhibited several favorable properties for gene delivery, including protection of nucleic acids from enzymatic degradation, stable colloids in serum, and redoxresponsive pDNA release. As a potential MR imaging agent, PSPIO displayed good magnetization (28.3 emu/g) and dose-dependent T2-weighted imaging contrast (R2 = 291.1 s−1 mM−1) in vitro. The use of redox-sensitive SSPEI polymer contributed to much lower cytotoxicity of PSPIO compared to nondegradable bPEI25k. In vitro transfection efficiency of PSPIO was significantly enhanced under an external magnetic field. In the presence of serum, PSPIO exhibited higher transgene expression than SSPEI or bPEI25k polymer on mouse glioma (ALTS1C1) or human prostate cancer (PC3) cell lines. Taken together, it is demonstrated that PSPIO possess great potential for cancer gene therapy and molecular imaging.

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(i.e., bPEI 25 kDa)11−16 has been widely utilized for SPIO functionalization. HMW PEI interacts with nucleic acids to form nanoparticles, which exhibit excellent cellular uptake efficiency. Taking advantages of the proton sponge effect, PEI promotes the nucleic acids escape from endosome for subsequent transgene expression. Meanwhile, the potential cytotoxic17−19 and host immunogenic effects20 associated with nondegradable cationic polymers are of major concerns. To tackle this challenge, we proposed a novel magnetofection system utilizing the cationic redox-sensitive PEI (SSPEI) aiming to achieve safe and efficient gene delivery. In comparison to nondegradable cationic polymers, SSPEI exhibits comparable transfection efficiency but much lower cytotoxicity.21,22 SSPEI can be conveniently prepared by performing the polymerization using disulfidecontaining linkers or direct oxidation.23 The redox-sensitive disulfide backbone of SSPEI allows minimal extracellular leakage and maximal intracellular release of nucleic acids for efficient transgene expression. To prepare PSPIO, the hydrophilic SSPEI was assembled with hydrophobic SPIO via ligand exchange process.24 The size, morphology, and surface potential of PSPIO were characterized using ZetaSizer and transmission electron microscope (TEM). The magnetism, T2 relaxivity and cell MR imaging of PSPIO were determined by SQUID and 7T MRI respectively. PSPIO was further studied for its pDNA interacting ability and the

INTRODUCTION Gene therapy has emerged as an advanced technique for disease treatment by applying functional nucleic acids such as plasmid DNA (pDNA),1 antisense oligonucleotides,2 or small interference RNA (siRNA)3,4 to correct specific molecular targets vital for disease development. Even though viral gene carriers have shown promising gene delivery performance; however, their clinical uses are still restricted by safety concerns.5 As a result, development of effective nonviral gene carriers has gained increasing attention on gene therapy in recent decades. Superparamagnetic iron oxide nanoparticles (SPIO) are an important class of multifunctional nanomaterial useful for various biomedical applications such as drug/gene delivery, magnetic resonance imaging (MRI), magnetic targeting, and magnetic hyperthermia therapy. SPIO can be synthesized in large quantity using coprecipitation or thermal decomposition methods. SPIO-based magnetofection is recognized as an effective gene delivery method in vitro and in vivo.6−8 For an ordinary magnetofection setup, nucleic acids are absorbed to the cationic SPIO-based carriers via electrostatic interactions. Under an external magnetic field, the induced magnetization in SPIO facilitates the contact of SPIO−nucleic acids complexes with the cells resulting in enhanced gene delivery efficiency.9,10 In the absence of a magnetic field, the unmagnetized SPIO is well-dispersed and suitable for injection. To construct effective gene carriers for magnetofection, it is required to functionalize SPIO with cationic materials capable of nucleic acids binding and controlled release. Among various cationic materials, high-molecular-weight (MW) polyethylenimine (HMW PEI) © 2015 American Chemical Society

Received: April 2, 2015 Revised: May 11, 2015 Published: May 19, 2015 6523

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nanomaterials. The excess solution was wiped off by filter paper and the samples were then subjected to drying in the oven overnight before examination by TEM (JEOL JEM-1200EX II, Japan). The hydrodynamic size and surface potential were measured using a Zetasizer Nano Series (Malvern, UK). The magnetism and hysteresis loop of PSPIO were examined by SQUID at 300 K (Quantum Design MPMS-XL, USA). The T2 relaxativity and T2-weighted imaging were studied by 7T MRI system (Bruker biospec 70/30 MRI, USA). The crystal structure of the synthesized SPIO was examined by Bruker D8 Advance X-ray diffractometer (Bruker, Karlsruhe, Germany). Gel Retardation Assay. Naked pDNA (200 ng) or PSPIO/pDNA (w/w ratio at 1, 4, 8, 12, and 16) was prepared as described above. The nanocomplexes were then mixed with O’ SAFE Red 6X Loading Dye (Omics Bio) and subjected to 0.9% agarose gel electrophoresis. DNase Protection Test. PSPIO/pDNA nanocomplex (w/w ratio = 8 containing 100 ng pDNA) or naked pDNA (100 ng) was treated with 20 mU DNase I (Worthington) in reaction buffer (10 mM Tris, 0.5 mM CaCl2, 2.5 mM, MgCl2, pH = 7.6) at 37 °C at total volume of 10 μL. At each incubation time (30, 60, 90, and 120 min), 5 μL of stop buffer (100 mM EDTA) was added into both PSPIO/pDNA and naked pDNA, which were then heated to 75 °C for 10 min to inactivate DNase I. Heparin was added to the mixture to dissociate pDNA from PSPIO at room temperature for 4 h. After that, the free pDNA was mixed with O’ SAFE Red 6X Loading Dye and subjected to 0.9% agarose gel electrophoresis as described above. Redox-Sensitive pDNA Release Test. The PSPIO/pDNA nanocomplex (w/w ratio = 8 containing 100 ng pDNA) was treated with or without 10 mM GSH at 37 °C for 24 h. The SPIO or digested SSPEI was then separated by centrifuged (15 000 × g, 10 min). The pDNA from each compartment was dissociated by heparin at room temperature for 4 h. Eventually, the mixtures were mixed with O’ SAFE Red 6X Loading Dye and subjected to 0.9% agarose gel electrophoresis as described above. The percentage of released pDNA in supernatant was quantitatively analyzed using a spectrophotometer (Nanodrop 2000, Thermo Scientific, Wilmington, USA). Cell Culture. HEK293T, ALTS1C1, and PC3 cells were cultured in DMEM supplemented with 10% FBS and 1× P/S maintained at 37 °C and 5% CO2 atmosphere. The hMSCs (FIRDI, Taiwan) were cultured in α-MEM supplemented with 20% FBS and 1× P/S at 37 °C and 5% CO2 atmosphere. As the cell confluency reached 80%, the cells were subcultured at a split ratio of 1:3. Cellular Uptake. 6 ×104 ALTS1C1 cells were seeded in 24-well plates and cultured at 37 °C overnight. After the cell confluency reached 60%, the cells were rinsed with PBS and replace with 25% FBS containing DMEM. PSPIO/pDNA nanocomplexes (in 5% glucose) at w/w ratio of 8 were added to the cells with or without magnet placing under a 24-well plate for 15 min. After that, the cells were washed twice with PBS, fixed by 4% paraformaldehyde in PBS for 10 min, and then stained by 10% hexacyanoferrate in 20% hydroxyl chloride solution for 20 min at room temperature. The cells were washed 3 times with deionized water before taking the cell staining images using Zeiss Axio Observer D1 (Carl Zeiss, Oberkochen, Germany). In Vitro Cytotoxicity. The cytotoxicity of PSPIO was determined by MTT assay. 5 × 104 hMSCs was seeded in 24-well plates and culture at 37 °C overnight. The hMSCs were treated with PSPIO/ pDNA (in 5% glucose) at different w/w ratios or bPEI25k at N/P ratio of 5 (1 μg pDNA per well) in serum free medium for 4 h. The cells were then washed with PBS, replaced with fresh medium, and incubated for another 24 or 48 h. At that time, cells were washed with PBS and replaced with fresh medium contain 5 μg/mL MTT to incubate for another 4 h. The supernatant was aspirated and the formazan was dissolved by DMSO. The supernatant was then transferred into 96-well plate, and the absorbance read at wavelength of 570 nm by microplate reader (infinite M200; Tecan, Austria). T2 Weighted Images. T2 weighted imaging of PSPIO or cells was performed by 7T MR imaging system (Bruker biospec 70/30 MRI, USA). Different ferric iron concentrations of PSPIO (1, 0.5, 0.25, 0.125, and 0.0625 mM Fe) in deionized water were deposited into 250 μL PCR tubes. For cell phantom images, 7 × 104 ALTS1C1 cells

resultant PSPIO/pDNA complexes were tested for the colloidal stability in various environments. The in vitro magnetofection efficiency of PSPIO was studied on mouse glioma cell line (ALTS1C1) and human prostate cancer line (PC3).

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EXPERIMENTAL SECTION

Materials. Iron(III) chloride, 3,3′-dithiodipropanoic acid (DTPA), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide. HCl (EDC), N-hydroxysuccinimide (NHS), trioctylphosphine oxide (TOPO), and paraformaldehyde were purchased from Alfa Aesar (Ward Hill, MA, USA). Polyethylenimine (800 Da or 25 kDa) and hexacyanoferrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oleic acid was purchased from SHOWA (Tokyo, Japan). Dialysis membrane (MWCO 3.5 kDa) was purchased from Spectrum Laboratories (Rancho Dominguez, CA, USA). A luciferase assay was purchased from Promega (Madison, WI, USA) and a bicinchoninic acid (BCA) protein assay reagent kit was purchased from Pierce Chemical Co (Rockford, IL, USA). Amiloride, chlorpromazine, and β-cyclodextrin were purchase from TCI (Tokyo, Japan). Sodium oleate was purchased from Nihon Shiyaku Reagent (Tokyo, Japan). MTT and ampicillin were purchased from MDBio, Inc. (Taipei, Taiwan). pcDNA3.Luciferase plasmid DNA was constructed by cloning luciferase gene from pGL3-basic (Promega) to pcDNA3 (Invitrogen) vector under control of the CMV promoter. The pDNA were amplified in DH5-α competent cell and purified by NucleoBond Xtra Midi EF Kit (Macherey Nagel, Düren, Germany). Dulbecco’s Modified Eagles’ Medium (DMEM), Minimum Essential Mediumalpha (α-MEM), and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Gibco (Carlsbad, CA, USA). Penicillin− streptomycin (P/S) and fetal bovine serum (FBS) were purchased from Hyclone (Logan, UT, USA). Synthesis of Redox-Sensitive Polyethylenimine (SSPEI). DTPA (1 mmol), 800 Da PEI (1 mmol), EDC (2.5 mmol), and NHS (2.5 mmol) were dissolved in 2.5 mL DMSO and stirred for 3 days at room temperature. The viscous reactant were purified by dialysis (3.4k cutoff) in DI water for 3 days and then lyophilized before further use. Synthesis of Superparamagnetic Iron Oxide Nanoparticles. The iron oxide nanoparticles were synthesized according to the procedure developed by Hyeon et al. In brief, iron oleate complex (2 mmol) was dissolved in 1-octadecene (10 mL) in the presence of oleic acid (0.5 mmol) and trioctylphosphine oxide (0.4 mmol). The mixture was heated to 320 °C and kept at that temperature for 2 h. After cooling down to room temperature, the mixture was washed with 4 mL hexane/ethanol (1:1) mixture and then collected by magnetic separation. The nanoparticles were resuspended in chloroform or hexane before further use. Synthesis of PSPIO with Ligand Exchange Process. Ten mg SPIO in 1 mL chloroform was mixed with 100 mg SSPEI, dissolved in 10 mL of DMSO/MeOH (1:1). The mixture were subjected to water bath sonication for 6 h. The formed PSPIO nanocomplex was collected by magnet attraction, then washed with 5 mL DI H2O 3 times, and the remaining organic solvent was removed by rotavap. PSPIO was reconstituted in 10 mL DI H2O containing 100 mg SSPEI and then subjected to a second ligand exchange. After two ligand exchange treatments, the PSPIO was collected by magnet and washed with DI H2O three times to remove free SSPEI. The total iron concentration of PSPIO was determined by the potassium thiocyanate method.25 Preparation of PSPIO/pDNA. The PSPIO/pDNA complexes were prepared by adding PSPIO in deionized water into pDNA in same volume. The mixture was vortexed for 15 s and incubated at room temperature for another 20 min to obtain PSPIO/pDNA. For in vitro experiments, the PSPIO/pDNA contained 5% glucose in the preparation steps. Physicochemical Characterizations of SPIO, PSPIO, and PSPIO/pDNA Nanocompelxes. The morphologies of SPIO, PSPIO, and PSPIO/pDNA were examined by TEM. SPIO, PSPIO, and PSPIO/pDNA were prepared in hexane or deionized water, respectively, and each sample was dropped on the carbon-coated copper grid, which was held at room temperature for 4 h to sediment 6524

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Langmuir were placed in each well in a 24-well plate and culture overnight. When the cell confluency reached 70%, the cells were rinsed with PBS and incubated with PSPIO/pDNA at w/w ratio of 8:1 for 15 min with the aid of an external magnet attraction in 25% FBS/DMEM before washing with PBS twice and trypsinization. Different amounts of cells were then fixed with 0.5% agarose and placed into 250 μL PCR tubes. A multislice multiecho (MSME)-T2 map pulse sequence, with static TR (6000 ms) and 32 fitted echoes in 11 ms intervals, was used to measure the spin−spin relaxation times (T2).26 The T2-weighted spin−echo sequence (TR/TE = 6000 ms/11 ms) was used for MR imaging. All samples were scanned by a fast gradient echo pulse sequence with the following parameters: TR/TE = 6000 ms/11 ms, matrix size = 256 × 256, FOV = 6 × 6 mm, and NEX = 3. In Vitro Magnetofection Assay. ALTS1C1 or PC3 cells were seeded at cell density of 6 × 104 cells per well in 24-well plates and cultured overnight. When the cell confluency reached 60%, the cell were rinsed with PBS and replace with 25% FBS containing DMEM. bPEI25k/pDNA at N/P ratio of 5, SSPEI/pDNA at N/P ratio of 60, or PSPIO/pDNA nanocomplexes containing 1 μg pDNA (pcDNA3.Luciferase) were added into cells with or without magnet placement under 24-well plates for 15 min. After that, the cells were washed with PBS twice and replaced with 10% FBS/DMEM. 48 h after transfection, the transgene expression was quantitated by luciferase assay using a luminance plate reader (infinite M200; Tecan, Austria) and normalized by BCA protein assay. Alternately, 72 h after transfection, the cell were rinsed with PBS and replaced with fresh medium. After 150 μg D-luciferin (Promega) was added to cells and allowed to react at room temperature for 5 min, the bioluminescence images were recorded by IVIS (Xenogen Caliper, Life Sciences, USA). For cellular uptake mechanism evaluation, ALTS1C1 cells were incubated with amiloride (inhibitor of macropinocytosis, 0.5 mM), chlorpromazine (inhibitor of clathrin-mediated endocytosis, 5 μg/mL), or β-cyclodextrin (inhibitor of caveolea-mediated endocytosis, 2 mM) in serum free DMEM for 30 min before adding the PSPIO/pDNA magnetoplex at w/w ratio of 8. After 4 h transfection without magnet attraction, cells were washed with PBS once and replaced with fresh 10% FBS/DMEM. 48 h after transfection, the transgene expression was

quantitated by luciferase assay and normalized by BCA protein assay as previously described. Statistics. Results of this study are presented as the mean and standard deviation of at least three independent measurements. All statistical evaluations were carried out with unpaired two-tailed Student’s t test. p-Value of less than 0.05 was considered significant (p < 0.05, * ; p < 0.01, ** ; p < 0.001, ***).

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RESULTS AND DISCUSSION Synthesis and Characterizations of PSPIO. The goal of this study is to develop a safe and effective gene delivery system by combining redox-sensitive SSPEI with SPIO. SSPEI was synthesized by coupling nontoxic low-molecular-weight PEI using a disulfide-containing cross-linker (DTPA) as previously described.23 The synthesized SSPEI was capable of efficiently transfecting various mammalian cell lines such as HEK293T, HeLa, and PC3 (Figure S1). Also, in contrast to bPEI25k, the synthesized SSPEI possessed negligible cytotoxicity (Figure S2). SPIO with an oleic acid (OA) shell was synthesized by thermal decomposition method,27 where the trioctylphosphine oxide (TOPO) was used as cosurfactant.24 The as-synthesized SPIO was characterized for its physical and chemical properties. By using dynamic light scattering (DLS), the size of SPIO was measured to be 15.8 nm (Figure 1d) which is similar to the size observed from TEM imaging (Figure 1a). X-ray diffractometry (XRD) was used to confirm the crystal structure of the synthesized SPIO to be γ-Fe3O4 (Figure S3). To prepare PSPIO, cationic SSPEI was used to substitute OA and TOPO from the surface of SPIO via a ligand exchange process. To promote the ligand exchange efficiency, DMSO was used as the reaction solvent due to its excellent solubility for both the hydrophilic SSPEI and the hydrophobic SPIO. The completion of the ligand exchange process was characterized by the transition from an initial muddy mixture to a transparent PSPIO solution.

Figure 1. TEM images of (a) SPIO, (b) PSPIO, and (c) PSPIO/pDNA nanocomplexes at weight ratio of 8:1. (d) Hydrodynamic size and surface potential measured by DLS. 6525

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Figure 2. Magnetic properties of SPIO and PSPIO. (a) Measurement of magnetization curve at 300 K. (b) PSPIO dispersed in deionized water before (left) and after (right) magnetic attraction for 5 min. (c) T2 relaxivity measurement.

Figure 3. Agarose gel electrophoresis. (a) Gel retardation of PSPIO/pDNA at different weight ratio. (b) DNase protection assay. Naked pDNA or PSPIO/pDNA was digested with DNase I at 37 °C for 30, 60, 90, or 120 min before electrophoresis analysis. NT: pDNA received no treatment.

the spin−spin relaxation time (T2) of PSPIO was measured by 7T MRI system. The calculated R2 (291.1 s−1 mM−1) of PSPIO (Figure 2c) was higher than the commercial MRI contrast agents such as Ferridex (63.5 s−1 mM−1) or Resovist (141 s−1 mM−1). Gel Retardation and DNase Protection Assay. The cellular uptake of pDNA by mammalian cells is an inefficient process due to the lack of adequate cellular uptake mechanism. Cationic gene carriers could condense anionic pDNA to form self-assembled nanoparticles, which could be efficiently taken up by cells via endocytosis. In PSPIO, SSPEI serves as a cationic scaffold for loading and delivering pDNA into cells. The pDNA condensation ability of PSPIO was studied using the gel retardation assay. The results (Figure 3a) showed that PSPIO was able to fully condense pDNA at or above the PSPIO/pDNA weight ratio of 4:1. The protection effect of PSPIO on pDNA was next evaluated by incubating the PSPIO/ pDNA nanocomplexes with DNase I at 37 °C for 2 h. The results (Figure 3b) showed that the integrity of pDNA was well preserved when it was complexed with PSPIO. The protection effect might be attributed to steric hindrance effect provided by PSPIO, which helped to prevent the contact of nucleic acid with the catalytical center of DNase I. Redox-Sensitive pDNA Release. After entering cells via endocytosis, PSPIO/pDNA nanocomplexes were anticipated to escape from endosome by proton sponge effect following by pDNA release via glutathione (GSH)-mediated SSPEI degradation. Glutathione (GSH) was chosen to mimic the intracellular redox environment. It has been previously reported31 that the intracellular GSH concentration ranges 2−10 mM, which is much higher than the extracellular GSH concentration (10−20 μM). To test this hypothesis, PSPIO/ pDNA nanocomplexes were treated with 10 mM GSH to

In the aqueous environment, PSPIO was highly positively charged and well dispersed by the repulsive forces from the protonated SSPEI. Physicochemical Characterizations of PSPIO. Size and surface potential are two important determinants for the transfection efficiency of nonviral gene carriers. Cationic polymer/ nucleic acid complexes could bind to the plasma membranebound heparin sulfate proteoglycan to trigger the endocytosis process.28−30 In this study, the size, surface potential, and PDI of PSPIO were 115 nm, +47.9 mV, and 0.142, respectively, as measured using a ZetaSizer (Figure 1d). TEM imaging was further used to study the morphology of PSPIO showing that PSPIO contained clusters of multiple SPIO assembled with SSPEI (Figure 1b). The cluster formation could be explained by the following two possibilities: (1) SSPEI may serve as crosslinkers to interbridge SPIOs into nanoclusters. (2) During the ligand exchange process, hydrophobic interaction-driven aggregation of SPIO occurred and resulted in the formation of nanoclusters. The formed PSPIO could be further used to assemble with pDNA via electrostatic interactions (Figure 1c). SPIO synthesized using thermal decomposition method is mainly composed of γ-Fe3O4 crystals with an ordered lattice structure that contributes to its strong superparamagnetivity. Then, the magnetization of the synthesized SPIO was measured using the superconducting quantum interference device (SQUID). Both OA-SPIO and PSPIO displayed a standard hysteresis loop for superparamagnetic materials (Figure 2a). The magnetism of SPIO and PSPIO was measured to be 48.6 emu/g and 28.3 emu/g, respectively. Lower magnetization of PSPIO was mainly attributed to the weight contribution from the SSPEI shell. The ferrofluidic property of PSPIO (Figure 2b) was observed under an external magnetic field. To evaluate the potential of using PSPIO as a MRI contrast agent, 6526

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size of PSPIO/pDNA (Figure 5a) remained stable (under 130 nm) during the incubation length (4 h). However, after incubating PSPIO/pDNA in serum free DMEM for 4 h, the particle size (Figure 5b) was increased from 200 nm to over 2000 nm possibly due to charge neutralization by adsorption of counterions from DMEM medium. Conversely, in 25% FBS-containing DMEM, the particle size of PSPIO/pDNA (Figure 5c) increased to approximately 600 nm at the weight ratio of 4:1. At higher weight ratios (8:1 and 12:1), the size remained small ( PSPIO (w/magnet) > SSPEI > PSPIO (w/o magnet) (Figure 10a). In the serum-containing condition, the transfection efficiency was PSPIO (w/magnet) > bPEI25k > SSPEI > PSPIO (w/o magnet) (Figure 10b). Regardless of the presence of serum, the transfection efficiency of PSPIO/pDNA was greatly enhanced by the application of an external magnetic field, suggesting successful magnetofection was attained. It is also noted that PSPIO/pDNA-mediated magnetofection exhibited higher transfection efficiency and lower cytotoxicity compared to the polymer control groups. Similarly, the magnetofection of SPIO was also demonstrated on the human prostate cancer cell line (PC3) (Figure S4). Furthermore, magnet-enhanced transfection was visualized on the ALTS1C1 cell line by 6528

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Figure 10. In vitro transfection of PSPIO/pDNA on ALTS1C1 cells. Transfection was performed under (a) DMEM or (b) 25% FBS/DMEM. 48 h after transfection, gene expression was quantitated by luciferase assay. (c) Bioluminescence images were acquired 72 h after transfection. Data represents the mean ± S.E.; n = 3.

Scheme 1. PSPIO-Mediated Gene Delivery into Mammalian Cells

main causes. First, an external magnetic field accelerated the contact of PSPIO/pDNA with the cell membrane to increase the chance of subsequent cellular uptake and transgene expression

observing higher intensity of bioluminescence images using IVIS (Figure 10c). The observed magnetically assisted transfection enhancement by PSPIO might be attributed to two 6529

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within a fixed incubation time length. Second, under serumcontaining condition, the stable and disperse nanosized PSPIO/ pDNA colloids (Figure 5c) were internalized into cells via clathrin- and caveolae-mediated endocytosis process.

CONCLUSION PSPIO composed of SPIO and SSPEI was successfully developed as a safe and efficient gene delivery system in this study (Scheme 1). The as-prepared PSPIO were positively charged nanoparticles which were capable of loading, condensing, and protecting pDNA from enzymatic digestion. The formed PSPIO/pDNA nanocomplexes displayed good colloidal stability in serum-containing environment. The magnetic PSPIO possess higher T2 relaxivity than the commercial MRI T2 contrast agents. Significantly, MR imaging can be detected in cells labeled with PSPIO in a dose dependent manner. Lower cytotoxicity was observed from PSPIO compared to its nondegradable counterpart (bPEI25k). The magnetofection of PSPIO was demonstrated on both mouse and human cancer cell lines in the serum-containing environment. Taking together, our findings suggest PSPIO as a potential carrier system for magnetofection. ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S4, which represent the evaluation of SSPEI transfection efficiency on various human cell lines, cytotoxicity of SSPEI compared to bPEI25k, the XRD pattern of as-synthesized SPIO and magnetofection on human prostate cancer line using PSPIO/pDNA. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.langmuir.5b01208.

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REFERENCES

(1) Won, Y. W.; Bull, D. A.; Kim, S. W. Functional polymers of gene delivery for treatment of myocardial infarct. J. Controlled Release 2014, 195, 110−119. (2) Hagigit, T.; Abdulrazik, M.; Valamanesh, F.; Behar-Cohen, F.; Benita, S. Ocular antisense oligonucleotide delivery by cationic nanoemulsion for improved treatment of ocular neovascularization: an in-vivo study in rats and mice. J. Controlled Release 2012, 160, 225− 231. (3) Son, S.; Nam, J.; Kim, J.; Kim, S.; Kim, W. J. i-motif-driven Au nanomachines in programmed siRNA delivery for gene-silencing and photothermal ablation. ACS Nano 2014, 8, 5574−5584. (4) Ku, S. H.; Kim, K.; Choi, K.; Kim, S. H.; Kwon, I. C. Tumortargeting multifunctional nanoparticles for siRNA delivery: recent advances in cancer therapy. Adv. Healthcare Mater. 2014, 3, 1182− 1193. (5) Giacca, M.; Zacchigna, S. Virus-mediated gene delivery for human gene therapy. J. Controlled Release 2012, 161, 377−388. (6) Scherer, F.; Anton, M.; Schillinger, U.; Henke, J.; Bergemann, C.; Kruger, A.; Gansbacher, B.; Plank, C. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther. 2002, 9, 102−109. (7) Plank, C.; Schillinger, U.; Scherer, F.; Bergemann, C.; Remy, J. S.; Krotz, F.; Anton, M.; Lausier, J.; Rosenecker, J. The magnetofection method: using magnetic force to enhance gene delivery. Biol. Chem. 2003, 384, 737−747. (8) Mykhaylyk, O.; Antequera, Y. S.; Vlaskou, D.; Plank, C. Generation of magnetic nonviral gene transfer agents and magnetofection in vitro. Nat. Protoc. 2007, 2, 2391−2411. (9) Dobson, J. Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery. Gene Ther. 2006, 13, 283−287. (10) Plank, C.; Zelphati, O.; Mykhaylyk, O. Magnetically enhanced nucleic acid delivery. Ten years of magnetofection-progress and prospects. Adv. Drug Delivery Rev. 2011, 63, 1300−1331. (11) Sun, S. L.; Lo, Y. L.; Chen, H. Y.; Wang, L. F. Hybrid polyethylenimine and polyacrylic acid-bound iron oxide as a magnetoplex for gene delivery. Langmuir 2012, 28, 3542−3552. (12) Zhou, Y.; Tang, Z.; Shi, C.; Shi, S.; Qian, Z.; Zhou, S. Polyethylenimine functionalized magnetic nanoparticles as a potential non-viral vector for gene delivery. J. Mater. Sci.: Mater. Med. 2012, 23, 2697−2708. (13) Wang, X.; Zhou, L.; Ma, Y.; Li, X.; Gu, H. Control of aggregate size of polyethyleneimine-coated magnetic nanoparticles for magnetofection. Nano Res. 2010, 2, 365−372. (14) Ota, S.; Takahashi, Y.; Tomitaka, A.; Yamada, T.; Kami, D.; Watanabe, M.; Takemura, Y. Transfection efficiency influenced by aggregation of DNA/polyethylenimine max/magnetic nanoparticle complexes. J. Nanopart. Res. 2013, 15, 1653−1664. (15) Liu, W.-M.; Xue, Y.-N.; Peng, N.; He, W.-T.; Zhuo, R.-X.; Huang, S.-W. Dendrimer modified magnetic iron oxide nanoparticle/ DNA/PEI ternary magnetoplexes: a novel strategy for magnetofection. J. Mater. Chem. 2011, 21, 13306−13315. (16) Kami, D.; Takeda, S.; Makino, H.; Toyoda, M.; Itakura, Y.; Gojo, S.; Kyo, S.; Umezawa, A.; Watanabe, M. Efficient transfection method using deacylated polyethylenimine-coated magnetic nanoparticles. J. Artif. Organs 2011, 14, 215−222. (17) Moghimi, S. M.; Symonds, P.; Murray, J. C.; Hunter, A. C.; Debska, G.; Szewczyk, A. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol. Ther. 2005, 11, 990−995. (18) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Controlled Release 2006, 114, 100−109. (19) Parhamifar, L.; Larsen, A. K.; Hunter, A. C.; Andresen, T. L.; Moghimi, S. M. Polycation cytotoxicity: a delicate matter for nucleic acid therapyfocus on polyethylenimine. Soft Matter. 2010, 6, 4001− 4009. (20) Chang, C. W.; Choi, D.; Kim, W. J.; Yockman, J. W.; Christensen, L. V.; Kim, Y. H.; Kim, S. W. Non-ionic amphiphilic

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 886-3-5715131 ext. 35531. Fax: 886-3-5718649. Author Contributions

R.Y. Huang contributed to experiment design, acquisition and analysis of data and drafting of manuscript. P.H. Chiang, W.C. Hsiao, and C.C. Chuang contributed to experiment design and acquisition of data. C.W. Chang contributed to study conception and design, interpretation of data and critical revision of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by National Health Research Institutes of Taiwan (NHRI-EX103-10221EC), Ministry of Science and Technology of Taiwan (102-2113-M007-006-MY2), and National Tsing Hua University (104N2046E1/104N2732E1). We thank Ms. C.-Y. Chien of Ministry of Science and Technology (National Taiwan University) for the assistance in TEM experiments and to 7T animal MRI Core Lab of the Neurobiology and Cognitive Science Center, National Taiwan University for technical and facility support. The magnetization was measured by using SQUID magnetometer (MPMS XL-7) at the National Chiao Tung University. We also thank Prof. C. S. Chiang (National Tsing Hua University) for providing the ALTS1C1 cells. 6530

DOI: 10.1021/acs.langmuir.5b01208 Langmuir 2015, 31, 6523−6531

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Langmuir biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. J. Controlled Release 2007, 118, 245− 253. (21) Son, S.; Namgung, R.; Kim, J.; Singha, K.; Kim, W. J. Bioreducible polymers for gene silencing and delivery. Acc. Chem. Res. 2012, 45, 1100−1112. (22) Cheng, R.; Feng, F.; Meng, F.; Deng, C.; Feijen, J.; Zhong, Z. Glutathione-responsive nano-vehicles as a promising platform for targeted intracellular drug and gene delivery. J. Controlled Release 2011, 152, 2−12. (23) Xia, W.; Wang, P.; Lin, C.; Li, Z.; Gao, X.; Wang, G.; Zhao, X. Bioreducible polyethylenimine-delivered siRNA targeting human telomerase reverse transcriptase inhibits HepG2 cell growth in vitro and in vivo. J. Controlled Release 2012, 157, 427−436. (24) Xu, Y.; Qin, Y.; Palchoudhury, S.; Bao, Y. Water-soluble iron oxide nanoparticles with high stability and selective surface functionality. Langmuir 2011, 27, 8990−8997. (25) Gupta, A. K.; Gupta, M. Cytotoxicity suppression and cellular uptake enhancement of surface modified magnetic nanoparticles. Biomaterials 2005, 26, 1565−1573. (26) Schultz-Sikma, E. A.; Joshi, H. M.; Ma, Q.; Macrenaris, K. W.; Eckermann, A. L.; Dravid, V. P.; Meade, T. J. Probing the chemical stability of mixed ferrites: implications for MR contrast agent design. Chem. Mater. 2011, 23, 2657−2664. (27) Park, J.; An, K.; Hwang, Y.; Park, J. G.; Noh, H. J.; Kim, J. Y.; Park, J. H.; Hwang, N. M.; Hyeon, T. Ultra-large-scale syntheses of monodisperse nanocrystals. Nat. Mater. 2004, 3, 891−895. (28) Christianson, H. C.; Belting, M. Heparan sulfate proteoglycan as a cell-surface endocytosis receptor. Matrix Biol. 2014, 35, 51−55. (29) Payne, C. K.; Jones, S. A.; Chen, C.; Zhuang, X. W. Internalization and trafficking of cell surface proteoglycans and proteoglycan-binding ligands. Traffic 2007, 8, 389−401. (30) Hess, G. T.; Humphries, W. H. t.; Fay, N. C.; Payne, C. K. Cellular binding, motion, and internalization of synthetic gene delivery polymers. Biochim. Biophys. Acta 2007, 1773, 1583−1588. (31) Schafer, F. Q.; Buettner, G. R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 2001, 30, 1191−1212. (32) Ruponen, M.; Honkakoski, P.; Tammi, M.; Urtti, A. Cell-surface glycosaminoglycans inhibit cation-mediated gene transfer. J. Gene Med. 2004, 6, 405−414. (33) Ruponen, M.; Honkakoski, P.; Ronkko, S.; Pelkonen, J.; Tammi, M.; Urtti, A. Extracellular and intracellular barriers in non-viral gene delivery. J. Controlled Release 2003, 93, 213−217. (34) Huth, S.; Lausier, J.; Gersting, S. W.; Rudolph, C.; Plank, C.; Welsch, U.; Rosenecker, J. Insights into the mechanism of magnetofection using PEI-based magnetofectins for gene transfer. J. Gene Med. 2004, 6, 923−936.

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DOI: 10.1021/acs.langmuir.5b01208 Langmuir 2015, 31, 6523−6531