Multifunctional Magnetic Nanoparticles Modified with

Apr 23, 2013 - Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro .... toluene were purchased from Duksan Pure Chemicals (Kor...
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Multifunctional Magnetic Nanoparticles Modified with Polyethylenimine and Folic Acid for Biomedical Theranostics Hyunhee Yoo,†,§ Seung-Kwan Moon,‡,§ Taewon Hwang,† Yong Seok Kim,† Joo-Hwan Kim,‡ Sung-Wook Choi,*,‡ and Jung Hyun Kim*,† †

Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Republic of Korea ‡ Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea ABSTRACT: This paper describes the preparation of magnetic nanoparticles modified with polyethylenimine (PEI)−folic acid (PF) conjugate and their potential biomedical applications. Magnetic nanoparticles modified with (3-(2-aminoethylamino)propyltrimethoxysilane) (AEAPS) were first prepared using a ligand exchange method to provide biocompatibility and hydrophilicity, and further conjugated with PF to carry gene and enhance specific uptake into cancer cells. We demonstrated the feasibility of the multifunctional magnetic nanoparticles as contrast agents in magnetic resonance imaging (MRI) and as gene carriers for gene delivery. In vitro results revealed that the cytotoxicity of the multifunctional magnetic nanoparticles was lower compared to that of pristine magnetic nanoparticles. Furthermore, we demonstrated the specific uptake of the magnetic nanoparticles modified with PF to KB cells using WI-38 cells as comparison by confocal microscopy. The PF-modified magnetic nanoparticles can potentially be employed as theranostic nanoplatforms for targeted gene delivery to cancer cells and simultaneous magnetic resonance imaging.

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hyperthermia, and magnetic field-assisted radionuclide therapy.7 Most of the reported multifunctional nanoparticles are limited to the combination of specific cellular uptake, MRI, and/or drug delivery. In addition, these nanoparticles tend to easily form agglomerates because of their high surface area-tovolume ratio. Furthermore, naked magnetic nanoparticles could be oxidized due to the fact that they are chemically highly active, leading to reduction in magnetism and dispersibility in media. Therefore, grafting or coating with organic or inorganic species onto the surface of the magnetic nanoparticles were often used for the protection of the magnetic nanoparticles.8 To address these issues, we employed PEI with a high molecular weight and folic acid for modification of magnetic nanoparticles and evaluated their magnetic property and magnetic resonance signal intensity for magnetic resonance imaging (MRI). PEI on the surface of magnetic nanoparticles may enhance colloidal stability, lower cell toxicity, and provide a lot of biding sites for genes and dyes. In addition, their cytotoxicity and specific cellular uptake in KB cells (a human nasopharyngeal carcinoma cell line) was investigated compared to WI-38 cells (a normal embryonic human diploid cell line). Furthermore, their potential as gene carriers was also demonstrated. We believe that the multifunctional magnetic

INTRODUCTION Theranostics is a promising approach based on the integration of therapy and diagnostic imaging for individualized treatment. This concept is useful for designing nanoparticles and enhancing their functionality. Recent advances in nanoparticle technology have led to the demonstration and realization of theranostics. The ideal theranostic nanoparticles should meet many of the requirements, including imaging sensitivity, accuracy of targeting, and delivery of therapeutic agents (e.g., drug, protein, or gene). In general, nanoparticles can be taken up via fluid phase endocytosis, receptor-mediated endocytosis, or phagocytosis.1 Therefore, surface-modified nanoparticles with functional molecules (e.g., monoclonal antibodies and folic acid) are considered as attractive materials for the specific cellular uptake.2 Among many functional molecules, folic acid has been employed to demonstrate preferential cellular uptake in cancer tissue because its receptor is overexpressed on the surface of cancer cells.3 Polyethylenimine (PEI) as a synthetic cationic polymer has a high efficiency for gene delivery due to its unique “proton sponge effect”.4 It is commonly used to modify nanoparticles positively for gene delivery.5 Iron oxide magnetic nanoparticles with a diameter of approximately 20 nm can be either paramagnetic or superparamagnetic.6 The superparamagnetic nanoparticles have been widely used for in vivo biomedical applications, including contrast-enhanced magnetic resonance imaging, tissue-specific release of therapeutic agents, © XXXX American Chemical Society

Received: December 27, 2012 Revised: April 17, 2013

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nanoparticles. TEM specimens were prepared by aspirating samples onto a copper EM grid. The average diameter of the magnetic nanoparticles was calculated from TEM images by analyzing at least 200 nanoparticles for each sample using ImageJ software (National Institutes of Health, USA). The hydrodynamic size and zeta potential of the magnetic nanoparticles were measured using a NanoS Zeta Sizer (Malvern Instruments) just after sonication for 10 s. The average particle sizes with error ranges were obtained from three measurements of each sample. Infrared spectra were recorded by a FT-IR spectrometer (TENSOR27, BRUKER, Netherlands), and X-ray diffraction (XRD) data were collected on a Shimadzu XD-D1 X-ray diffractometer employing Cu Kα radiation at 30 kV and 30 mA. Magnetization loops were measured at room temperature using a physical property measurement system (MPMS XL7 Evercool Dewar Control Option, Quantum Design). MRI. The MRI experiments were performed with a 1.5 T clinical MRI instrument equipped with a micro-47 surface coil (Intera; Philips Medical Systems, Best, Netherlands). The T2 weights of the OAcoated magnetic nanoparticles and PF-modified magnetic nanoparticles were measured using the Carr−Purcell−Meiboom−Gill (CPMG) sequence at room temperature measure (RT = 10 s, 32 echoes with 12 ms even echo space, number of acquisitions = 1, point resolution of 156 × 156 mm, and section thickness = 0.6 mm). The following parameters were adopted for the acquisition of T2-weighted MRIs of the aqueous dispersion of OA-coated magnetic nanoparticles and PF-modified magnetic nanoparticles: resolution of 234 × 234 mm, section thickness 2.0 mm, TE = 60 ms, RT = 4000 ms, and number of acquisitions = 1.10 Cellular Uptake Studies. KB and WI-38 cells were purchased from the Korean Cell Line Bank and cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin in a humidified atmosphere containing 5% CO2 at 37 °C. To observe the cellular uptake, the different types of magnetic nanoparticles were tagged with fluorescent dye, RITC, using a procedure similar to that normally employed for RITC-coupling to protein molecules.11 AEAPS-modified and PF-modified magnetic nanoparticles (1 mg) were separately suspended in sodium carbonate buffer (1 mL, 0.5 M) at pH 9.5 to ensure the deprotonation of the amine groups on the AEAPS and PF conjugates. RITC solution (10 mg/mL in DMSO, 0.08 mL) was added to each suspension of magnetic nanoparticles and the mixture was covered with aluminum foil at room temperature overnight. The RITC-tagged magnetic nanoparticles were washed with distilled water and centrifuged until no further fluorescence was observed in the supernatant.9 Each cell line (KB and WI-38, 1 × 105 cells/well) was seeded on 6well plates and the plates were covered with autoclaved cover glass, followed by incubation overnight. The different types of RITC-labeled magnetic nanoparticles (100 μL, 0.25 mg/mL) were added to the cultured cells, incubated for 4 h, and subsequently rinsed with PBS three times, followed by adding paraformaldehyde solution (2 mL, 4%). After 15 min, the paraformaldehyde solution was removed and the cells were rinsed with PBS three times. The cells/magnetic nanoparticles complexes were examined by using a confocal laser scanning microscope (LSM 510 META, Carl Zeiss Inc.).10,11 Cytotoxicity Experiments. The cytotoxicity of the magnetic nanoparticles against KB and WI-38 cells was investigated using MTS assay. The cells were cultured in a 5% CO2 atmosphere at 37 °C. Each type of cells was seeded onto 96-well plates at a density of 1 × 105 cells/well and incubated in complete RPMI1640 for 24 h. The media was replaced with serum-free RPMI1640 and the different types of the magnetic nanoparticles were added to each well. After 3 days, serumfree media was discarded and MTS solution (20 μL) was added to each well, followed by incubation for 72 h. The absorbance was measured by an ELISA plate reader (VersaMax, Molecular devices) at a wavelength of 590 nm.

nanoparticles with capabilities of specific cellular uptake, MRI, fluorescence imaging, and gene delivery have great potential for theranostic applications.

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EXPERIMENTAL SECTION

Materials. Polyethylenimine (PEI, Mw 1.8 or 25 kDa), folic acid, ferrous sulfate hexahydrate, ferric chloride hexahydrate, sodium chloride (NaCl), oleic acid (OA), rhodamine B isothiocyanate (RITC), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Aldrich Chemical (USA). 3-(2-Aminoethylamino) propyltrimethoxysilane (AEAPS) was purchased from TCI America. Ammonium hydroxide solution (NH4OH), dimethyl sulfoxide (DMSO), and toluene were purchased from Duksan Pure Chemicals (Korea). Synthesis of PF Conjugates. Folic acid (1 mol), NHS (1.1 mol), EDC (1.1 mol), and PEI (5 mol) were dissolved in DMSO (50 mL). The reaction mixture was stirred overnight in darkness at room temperature. The solution containing the PF conjugates was dialyzed against distilled water for 3 days by using a dialysis membrane (MWCO 1000) and then freeze-dried. Synthesis of Magnetic Nanoparticles Modified with PF. Ferrous sulfate heptahydrate (FeSO4·7H2O, 2.35 g) and ferric chloride hexahydrate (FeCl3·6H2O, 4.1 g) were dissolved in distilled water (100 mL) with gentle stirring in a double jacketed reactor under nitrogen atmosphere; NH3H2O (25 wt %, 25 mL) was quickly poured into the aqueous solution at room temperature. Under vigorous stirring, OA (1 mL) was slowly dropped into the aqueous solution at 80 °C and the reaction was kept for 1 h. The obtained magnetic nanoparticles were dispersed in water after another 1 h. Afterward, the aqueous dispersion (50 mL) containing the magnetic nanoparticles was mixed with toluene (50 mL) at room temperature. Then, sodium chloride (0.2 g) was added to the mixture to induce the transfer of the magnetic nanoparticles from the water to the toluene phase. Finally, the toluene phase was collected and refluxed under nitrogen atmosphere to remove water, and the concentration of the magnetic nanoparticles in toluene was adjusted to 10 mg/mL. To exchange OA with AEAPS, AEAPS (0.1 mL), TEA (8 mL), toluene (20 mL), and the magnetic nanoparticles dispersed in toluene (10 mL) were poured into a 50-mL round-bottom flask. The mixture was stirred for 48 h at room temperature under nitrogen atmosphere. Afterward, petroleum ether (20 mL) was added to the mixture to precipitate the AEAPS-modified magnetic nanoparticles. The AEAPSmodified magnetic nanoparticles were collected, dried in vacuum, and redispersed in toluene, which was repeated five times. Finally, the dried magnetic nanoparticles were dispersed in water at a concentration of 1 mg/mL. For the reaction between the PF conjugates and AEAPS-modified magnetic nanoparticles, PF conjugates (0.05 mmol) were added to the aqueous dispersion (20 mL) containing the AEAPS-modified magnetic nanoparticles, together with NHS (1.1 mol) and EDC (1.1 mol). The mixture was stirred for 48 h at room temperature under nitrogen atmosphere. The resulting magnetic nanoparticles modified with PF conjugates were separated using a magnet and washed with water, which was repeated five times. Formation of the MNP-PF/DNA Complexes. Calf thymus DNA (approximately 1700 base pairs) was kindly donated from the Seoul National University (Republic of Korea). To quantify the DNAbinding capacity of the PF-modified magnetic nanoparticles with PEI of different Mw, PF-modified magnetic nanoparticles (20−80 μg per 1 mL) were mixed with 100 μg of DNA in deionized water (1 mL). The suspensions were incubated at room temperature under gentle rotation. After centrifugation, the amount of unbound DNA in the supernatant was determined by using an UV−vis spectrophotometer at 260 nm. The amount of DNA bound to the PF-modified magnetic nanoparticles was calculated by subtracting the amount of unbound DNA from the total amount of DNA.9 Characterization of Magnetic Nanoparticles. Transmission electron microscopy (TEM, JEM-2010(HC), 200 kV) was used to examine the morphology of the different types of magnetic

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RESULTS AND DISCUSSION Synthesis of PF-Modified Magnetic Nanoparticles. Figure 1a schematically shows the synthetic procedure for B

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modified magnetic nanoparticles had a large amount of amine groups on their surface compared to other previous magnetic nanoparticles, allowing for the further conjugation with rhodamine and gene without destroying colloidal stability. Therefore, the multifunctionality of the PF-modified magnetic nanoparticles can be extended to a specific cellular uptake, MRI, fluorescence imaging, and gene delivery. Characterization. The average hydrodynamic diameter of the PF-modified magnetic nanoparticles with 25 kDa of PEI was 14.8 ± 2.7 nm, which was slightly larger than that observed from TEM image. The presence of the PF layer on the surface of the PF-modified magnetic nanoparticles resulted in an increase of their hydrodynamic diameters. Figure 2 shows FT-

Figure 1. (a) Schematic diagram for the synthesis of PF-modified magnetic nanoparticles and TEM images of (b) AEAPS- and PFmodified magnetic nanoparticles with (c) Mw 1.8 and (d) 25 kDa of PEI. Insets (b, c, and d) are HR-TEM images and the arrows in the insets (c and d) indicate a thin layer of PF conjugate.

fabricating PF-modified magnetic nanoparticles. Folic acid was conjugated to PEI via well-known carbodiimide chemistry using EDC and NHS, obtaining PFI−folic acid conjugates (hereafter referred to as PF). For the synthesis of magnetic nanoparticles, an aqueous dispersion of Fe3O4 magnetic nanoparticles was prepared by coprecipitation method using OA as a surfactant. Afterward, OA molecules on the surface of the magnetic nanoparticles were replaced with hydrophilic AEAPS by ligand exchange method,10 yielding AEAPS-modified magnetic nanoparticles with a positive charge and enhanced hydrophilicity. Finally, PF conjugates were cross-linked to AEAPS on the surface of the magnetic nanoparticles via carbodiimide chemistry.12 Figure 1b shows TEM images of the different types of magnetic nanoparticles. The OA- and AEAPS-modified magnetic nanoparticles had average diameters of 10.1 ± 2.5 and 10.5 ± 2.2 nm, respectively. Even after PF conjugation onto the AEAPS-modified magnetic nanoparticles, their spherical morphologies were maintained but the diameter increased to 11.3 ± 2.6 nm due to the layer of the PF conjugate (Figure 1c and d). A PF layer can be clearly identified in the TEM images of the PF-modified magnetic nanoparticles. In addition, the lattice fringes in the insets correspond to a group of atomic planes within a single crystal of magnetic particles.13 The characteristic feature of the PF-modified magnetic nanoparticles lies on the decoration of PEI on the surface of the magnetic nanoparticles. Due to the high molecular weight of PEI, the PF-

Figure 2. FT-IR spectra of PEI, folic acid, PF conjugate, AEAPSmodified magnetic nanoparticles, and PF-modified magnetic nanoparticles.

IR spectra of PEI, folic acid, PF conjugate, AEAPS-modified magnetic nanoparticles, and PF-modified magnetic nanoparticles. Folic acid has two −COOH groups at the α and γ positions, which can act as linkers for covalent attachment. FTIR measurement showed that folic acid was grafted onto PEI chains through amide linkage. Upon grafting, strong absorption around 1657 cm−1 attributable to the amide linkage (amide, CO) appeared in the spectra of the PF conjugate.12 In addition, the PF conjugate showed strong and broad characteristic absorption of amines at 3400 cm−1 (NH2). The characteristic band of the phenyl ring from folic acid was shown at 1484 cm−1.14 Hydroxyl groups of OA on the OAcoated magnetic nanoparticles were reacted with methoxy of silane group in AEAPS and presented a capping ligand of covalent Fe−O−Si bond. The peaks at 1110, 804, and 471 cm−1 correspond to asymmetric stretching, symmetric stretching, and bending modes of Si−O−Si, respectively.15 The highresolution XRD pattern of PF-modified magnetic nanoparticles shows the inverse spinel structure of the iron oxide core (Figure 3).16 The XRD patterns can be indexed to be (2 2 0), (3 1 1), C

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modified magnetic nanoparticles (64 emu/g). This reduction in magnetic moment might be attributed to the existence of a magnetically dead layer on the surface of particles or a spinglass-like behavior of the surface of spins or canted spins.8 In addition, electron exchange between the ligand and surface atoms could also quench the magnetic moment.17 We evaluated the potential use of PF-modified magnetic nanoparticles as MRI agent. Figure 4b shows T2-weighted MRIs of the AEAPSand PF-modified magnetic nanoparticles at 1.5 T. MRI signal intensities diminished in all samples as the concentration of the magnetic nanoparticles decreased.18 The reduction in the MRI signal intensity corresponds to the results found in magnetic hysteresis. In fact, MRI signal intensity is strongly dependent on the concentration of the magnetic nanoparticles. Therefore, maximizing the concentration of magnetic nanoparticles taken up in target cells or tissues should be an attractive strategy for enhancing MRIs. This was our rationale to employ folic acid onto the surface of magnetic nanoparticles for MRI. Cytotoxicity Test. To evaluate the toxicity of the PFmodified magnetic nanoparticles (Mw 1.8 and 25 kDa of PEI), the viability of two different cell lines (KB and WI-38 cells) was examined at various concentrations of the magnetic nanoparticles. The MTS value when each type of cells was cultured without magnetic nanoparticles was set as 100%. As shown in Figure 5, the viabilities of KB and WI-38 cells tended to decrease as the concentration of the magnetic nanoparticles

Figure 3. XRD patterns of AEAPS- and PF-modified magnetic nanoparticles.

(4 2 2), (5 1 1), (4 0 0), and (4 4 0) planes of the typical Fe3O4 magnetic nanoparticles. All detected diffraction peaks could be attributed to the characteristic peaks of spinel magnetite (JCPDS card 85-1436).12b The γ-Fe2O3 magnetic nanoparticles can be characterized by two superlattice diffractions from (2 1 0) and (2 1 3) planes. Figure 4a shows superparamagnetic behaviors of the AEAPSand PF-modified magnetic nanoparticles at room temperature. The saturation magnetization of PF-modified magnetic nanoparticles (35 and 46 emu/g for 1.8 and 25 kDa of PEI, respectively) was found to be lower than that of the AEAPS-

Figure 4. (a) Magnetic hysteresis curves and (b) T2-weighted MRIs of AEAPS- and PF-modified magnetic nanoparticles with Mw 1.8 and 25 kDa of PEI.

Figure 5. Cytotoxicity test of AEAPS- and PF-modified magnetic nanoparticles with Mw 1.8 and 25 kDa of PEI using (a) KB and (b) WI-38 cells (n = 3). D

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column). A close observation of KB cells uptaking the PFmodified magnetic nanoparticles indicates that the confocal images were at the same depth-plane to the inside the cells because the nuclei were distinguishable as dark areas within KB cells. Therefore, it can be confirmed that most of the nanoparticles existed in the cytoplasm, not on the surface of the cells. Despite the lower MRI signal intensity of PF-modified magnetic nanoparticles compared to that of the other magnetic nanoparticles, the extremely high efficiency and specific uptake by KB cells were achieved by employment of PF conjugation onto the surface of magnetic nanoparticles. By taking Figures 5 and 6 into consideration, there was not much difference in cytotoxicity of the PF-modified magnetic nanoparticles between KB and WI-38 cells, although a large amount of the nanoparticles were uptaken by KB cells, not WI38 cells. There are previous reports that cell viability was not significantly decreased in some cases even after the cellular uptake of nanoparticles or substances.22 This result suggests that the uptake amount of nanoparticles was not the only factor for cell viability. Furthermore, to extend the function of magnetic nanoparticles to gene delivery, PEI with a high positive charge was employed onto the surface of magnetic nanoparticles in the form of PEI−folic acid conjugates (Figure 7). DNA from calf

increased. However, note that PF-modified magnetic nanoparticles exhibited lower cytotoxicity than the AEAPS-modified magnetic nanoparticles. In addition, the cytotoxicity of the PFmodified magnetic nanoparticles with PEI of high Mw (25 kDa) was almost close to 100% at a low concentration (0.25 mg/mL) and the same tendency was observed at other concentrations. This reduction in cytotoxicity might be attributed to the screening effect of PEI with a relatively low toxicity on the surface of the magnetic nanoparticles.4 Although PEI is, in general, known to have cytotoxicity, it was reported that its detrimental effects to cells were reduced when it wss bound to other molecules (e.g., DNA, folic acid).19 There should be a difference in the amount of cell uptake between KB and WI-38 cells. However, the observed cytotoxicity showed almost the same tendency because the cell viability generally decreases with respect to the amount of added nanoparticles or substances.20 Cellular Uptake. To investigate the cellular uptake, each type of cells (KB and WI-38 cells) was incubated for 4 h with the AEAPS-, PEI-, and PF-modified magnetic nanoparticles. KB cells overexpressing the folic acid receptor and WI-38 cells that do not overexpress the folic acid receptor were chosen to demonstrate selective targeting of PF-modified magnetic nanoparticles to folic acid receptor-positive cells.21 For visualization in confocal microscopy, RITC was coupled to the magnetic nanoparticles prior to the experiment. Figure 6

Figure 7. Plot of the amount of binding DNA as a function of the amount of PF-modified magnetic nanoparticles with Mw 1.8 and 25 kDa of PEI (n = 3).

thymus was used to examine the binding capacity. The average hydrodynamic diameter of the DNA-bound magnetic nanoparticles was increased to 72.4 ± 15.9 nm due to the relatively large size of DNA, suggesting that one DNA could be complexed with many magnetic nanoparticles and formed into clusters. However, the zeta potential was slightly reduced from +40.1 to +31.4 mV due to the binding of DNA on the surface of the magnetic nanoparticles. These results suggest a good colloidal stability of the PF-modified magnetic nanoparticles with DNA. It was found that the PF-modified magnetic nanoparticles with 25 kDa of PEI exhibited a superior binding capacity to that with 1.8 kDa of PEI, which is attributed to the large number of amine groups in PEI with a high molecular weight.23 The DNA binding capacity of the PFmodified magnetic nanoparticles was found to be about 61.77 and 86.12% DNA for PF-modified magnetic nanoparticles with Mw 1.8 and 25 kDa of PEI, respectively.

Figure 6. Representative confocal microscopy images of KB and WI38 cells after 4 h incubation at 37 °C with the AEAPS-, PEI-, and PFmodified magnetic nanoparticles containing RITC.

shows confocal microscopy images of the cell/magnetic nanoparticles complex. Little amounts of all types of magnetic nanoparticles was taken up by WI-38 cells. In contrast, a significant amount of PF-modified magnetic nanoparticles was only taken up by KB cells compared to the AEAPS- and PEImodified magnetic nanoparticles. It is worth pointing out that the shape of each cell observed by confocal microscopy (right column) was exactly matched to the optical images (left E

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(10) Lim, E. K.; Jang, E.; Kim, B.; Choi, J.; Lee, K.; Suh, J. S.; Huh, Y. M.; Haam, S. J. Mater. Chem. 2011, 21, 12473−12478. (11) Yim, H.; Jo, E. A.; Na, K. Macromol. Res. 2010, 18, 913−918. (12) (a) Liao, M. H.; Chen, D. H. J. Mater. Chem. 2002, 12, 3654− 3659. (b) Mohapatra, S.; Mallick, S.; Maiti, T.; Ghosh, S.; Pramanik, P. Nanotechnology 2007, 18, 385102. (13) Lou, L.; Yu, K.; Zhang, Z.; Li, B.; Zhu, J.; Wang, Y.; Huanga, R.; Zhu, Z. Nanoscale 2011, 3, 2315−2323. (14) Zhang, J.; Rana, S.; Srivastava, R.; Misra, R. Acta. Biomater. 2008, 4, 40−48. (15) Barick, K. C.; Bahadur, D. Bull. Mater. Sci. 2006, 29, 595−598. (16) Sun, Y.; Ding, X.; Zheng, Z.; Cheng, X.; Hu, X.; Peng, Y. Chem. Commun. 2006, 2765−2767. (17) Vanleeuwen, D. A.; Vanruitenbeek, J. M.; Dejongh, L. J.; Ceriotti, A.; Pacchioni, G.; Haberlen, O. D.; Rosch, N. Phys. Rev. Lett. 1994, 73, 1432−1435. (18) Bulte, J. W. M.; Douglas, T.; Witwer, B. Nat. Biotechnol. 2001, 19, 1141−1147. (19) Klemm, A. R.; Young, D.; Lloyd, J. B. Biochem. Pharmacol. 1998, 56, 41−46. (20) (a) Gupta, A. K.; Gupta, M. Biomaterials 2005, 26, 1565−1573. (b) Brunner, T. J.; Wick, P.; Manser, P.; Spohn, P.; Grass, R. N.; Limbach, L. K.; Bruinink, A.; Stark, W. J. Environ. Sci. Technol. 2006, 40, 4374−4381. (21) (a) Saula, J. M.; Annapragada, A.; Natarajanb, J. V.; Bellamkonda, R. V. J. Controlled Release 2003, 92, 49−67. (b) Sudimack, J.; Lee, R. J. Adv. Drug. Delivery Rev. 2000, 41, 147− 162. (c) Bhattacharyya, S.; Kudgus, R. A.; Bhattacharya, R.; Mukherjee, P. Pharm. Res. 2011, 28, 237−259. (d) Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M. Adv. Drug. Delivery Rev. 2008, 60, 1307− 1315. (22) (a) Arnida; Malugin, A.; Ghandehari, H. J. Appl. Toxicol. 2010, 30, 212−217. (b) Petri-Fink, A.; Steitz, B.; Finka, A.; Salaklang, J.; Hofmann, H. Eur. J. Pharm. Biopharm. 2008, 68, 129−137. (23) Suh, J.; Paik, H. J.; Hwang, B. K. Bioorg. Chem. 1994, 22, 318− 327.

CONCLUSIONS We have successfully fabricated multifunctional magnetic nanoparticles modified with PEI and folic acid. Iron oxide nanoparticles, PEI, and folic acid were utilized for achieving multifunctionality of MRI, gene delivery, and specific uptake by cancer cells, respectively. Despite the reduction in MR signal intensity due to PF conjugate, we still believe that the quality of MRI could be improved by maximizing the amount of magnetic nanoparticles taken up by the cells. The employment of a large amount of folic acid on the surface of nanoparticles could facilitate the cellular uptake for better MRI quality. However, the colloidal stability and size of magnetic nanoparticles should be properly taken into consideration for particle design. In the next step, our goal will be focused on in vivo animal study to demonstrate theranostics capable of subsequent MRI and gene therapy specific to cancer cells.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (S.-W.C.); [email protected]. kr (J.H.K.). Author Contributions §

H.Y. and S.-K.M. equally contributed to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported, in part, by Nano·Material Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2008-2002380 and 2012-0006227), the Research Grant funded by the Gyeonggi Regional Research Center (GRRC), and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0023064).

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REFERENCES

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