Clustered Magnetite Nanocrystals Cross-Linked with PEI for Efficient

Biomacromolecules 2011, 12, 457–465

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Clustered Magnetite Nanocrystals Cross-Linked with PEI for Efficient siRNA Delivery Ji Won Park, Ki Hyun Bae, Chunsoo Kim, and Tae Gwan Park* Department of Biological Sciences and Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea Received October 18, 2010; Revised Manuscript Received December 11, 2010

Magnetofection has been utilized as a powerful tool to enhance gene transfection efficiency via magnetic fieldenforced cellular transport processes. The accelerated accumulation of nucleic acid molecules by applying an external magnetic force enables the rapid and improved transduction efficiency. In this study, we developed magnetite nanocrystal clusters (PMNCs) cross-linked with polyethylenimine (PEI) to magnetically trigger intracellular delivery of small interfering RNA (siRNA). PMNCs were produced by cross-linked assembly of catechol-functionalized branched polyethylenimine (bPEI) around magnetite nanocrystals through an oil-in-water (O/W) emulsion and solvent evaporation method. The physical properties of PMNC were characterized by TEM, DLS, TSA, and FT-IR. Finely tuned formulation of clustered magnetite nanocrystals with controlled size and shape exhibited superior saturation of magnetization value. Magnetite nanocrystal clusters could form nanosized polyelectrolyte complexes with negatively charged siRNA molecules, enabling efficient delivery of siRNA into cells upon exposure to an external magnetic field within a short time. This study introduces a new class of magnetic nanomaterials that can be utilized for magnetically driven intracellular siRNA delivery.

Introduction In recent years, diverse inorganic nanomaterials based on gold, silver, iron oxide, and quantum dots have received significant attention as promising platform nanomaterials for efficient intracellular delivery of bioactive therapeutic macromolecules because of their tunable size and shape and unique optoelectronic properties.1 Particularly, superparamagnetic iron oxide nanoparticles have been extensively utilized in biomedical fields, ranging from the contrast agents for magnetic resonance imaging (MRI) to the carriers for magnetic drug/gene delivery and hyperthermic cancer treatment.2-5 For instance, magnetite nanoparticles conjugated with methotrexate, a powerful anticancer drug, exhibited significant apoptotic death of cancer cells and simultaneously detected the cells by their MRI contrast enhancement.6,7 Moreover, recent studies have attempted to create efficient drug delivery systems by combining iron oxide nanoparticles with magnetic forces.8 In this approach called “magnetofection”, an application of a magnetic field gradient rapidly concentrates the iron oxide nanoparticles coupled with therapeutic agents (e.g., drugs and genetic materials) to target cells or tissues, thereby providing site-specific drug targeting and retention.9-11 For effective magnetofection, it would be utmost important to produce iron oxide nanoparticles with an improved magnetic responsiveness. One approach for this purpose is to fabricate micrometer-sized magnetite particles; however, they tend to aggregate in aqueous solution due to the loss of the superparamagnetic properties. The other approach is to assemble iron oxide nanoparticles into a cluster. In contrast to the µm-sized particles, the clustering of magnetite nanoparticles greatly improved the magnetic responsiveness compared to individual nanoparticles, as well as maintained their superparamagnetism and dispersion stability.12-15 * To whom correspondence should be addressed. Tel.: +82-42-350-2621. Fax: +82-42-350-2610. E-mail: [email protected].

Small interfering RNA (siRNA), a class of double-stranded RNA molecules with 20-25 nucleotides in length, has been emerging as a promising nucleic acid therapeutic agent. Due to its superior ability to silence a target gene in a highly specific manner, siRNA has become a subject of growing interest to treat diverse malignant, infectious, and autoimmune diseases.16-18 Nonetheless, clinical use of siRNA has been restricted by its inherent instability in biological environments along with poor cellular uptake and endosomal escape. To overcome these limitations, various nonviral vectors including cationic polymers, peptides, and lipids have been extensively explored to improve the delivery efficiency and gene silencing effect of siRNA.19,20 For example, cationic polymers such as polyethylenimine (PEI) can form compact nanoscale polyelectrolyte complexes through electrostatic interactions with anionic siRNA molecules.21 These self-assembling polyelectrolyte complexes are able to facilitate the intracellular uptake of siRNA via an endocytosis pathway as well as to protect siRNA from degradation by nucleases in the bloodstream. To utilize siRNA for therapeutic applications, however, further improvements are still needed to achieve selective targeting of siRNA to desired cells or tissues, as well as to reduce cytotoxicity and immune reactions exerted by the cationic vectors. This study presents synthesis, characterization, and delivery applications of siRNA using a novel magnetofection platform based on surface functionalized magnetite nanoparticle clusters. For synthesis of these nanoparticle clusters, we utilized 3,4dihydroxy-L-phenylalanine (DOPA) as a robust anchor for surface immobilization of branched PEI onto the magnetite nanoparticles. DOPA is an unusual amino acid found within specialized adhesive foot proteins of marine blue mussels (Mytilus edulis).22 This amino acid is known to play an important role in the attachment of marine blue mussels on a wide range of organic and inorganic substrates. In particular, DOPA has been widely applied for anchoring small organic molecules or polymers on the surface of iron oxide nanoparticles

10.1021/bm101244j  2011 American Chemical Society Published on Web 12/29/2010

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because of its strong affinity to diverse metal oxides.23 In this study, DOPA-conjugated branched PEI (PEI-DOPA) was prepared and utilized as a cross-linker for spherical assembly of individual magnetite nanoparticles. The surface of the magnetite nanoparticle clusters was subsequently conjugated with poly(ethylene glycol) (PEG) for the formation of stable polyelectrolyte complexes in the serum conditions.24 The resultant PEG-functionalized magnetite nanoparticle clusters (PMNCs) having a highly cationic PEI shell layer were expected to condensate anionic siRNA into compact polyelectrolyte complexes. Moreover, PMNCs would exhibit a superior magnetoresponsible mobility compared to nonclustered magnetite nanoparticles and, thus, efficiently deliver the loaded siRNA into target cancer cells via magnetic force-assisted cellular transport processes. The size and structure of PMNCs were examined by using dynamic light scattering analysis and transmission electron microscopy. The extent of cellular uptake and gene silencing efficiencies were evaluated to demonstrate the feasibility of PMNCs for magnetically triggered intracellular delivery of siRNA.

Materials and Methods Materials. Branched polyethylenimine (PEI; average Mw ) 25000), hydrocaffeic acid, rhodamine B isothiocyanate, and Triton-X 100 were purchased from Sigma (St. Louis, MO). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). Methoxy-poly(ethylene glycol)succinimidyl-succinate (mPEG-SS; Mw ) 5000) was obtained from Sunbio Co. (Anyang, Korea). Green fluorescent protein (GFP) siRNA duplexes composed of sense (5′-AACUUCAGGGUCAGCUUGCdTdT3′) and antisense (5′-GCAAGCUGACCCUGAAGUUdTdT-3′) strands were obtained from Bioneer Co. (Daejeon, Korea). Human prostate carcinoma (PC-3) cells were acquired from Korean Cell Line Bank (Seoul, Korea), and PC-3 cells overexpressing GFP were donated from Samyang Corp. (Daejeon, Korea). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (MD, U.S.A.). Iron oxide nanoparticles (11 nm) were kindly donated from Professor Taeghwan Hyeon at Seoul National University, Korea. Synthesis of DOPA-Conjugated Branched PEI (PEI-DOPA). PEIDOPA was synthesized by conjugating a carboxylic acid group of hydrocaffeic acid (an analog of DOPA) to a primary amine group of bPEI via carbodiimide coupling chemistry. Hydrocaffeic acid (2.11 g, 11.6 mmol) dissolved in 5 mL of DMF was added to 5 mL of methanol containing EDC (4.44 g, 23.2 mmol). The resulting activated hydrocaffeic acid was subsequently reacted with branched PEI (2 g, 11.6 mmol) dissolved in 5 mL of methanol for 12 h at room temperature. The reactants were precipitated into cold diethyl ether twice, and then dialyzed against HCl solution (pH 4) for 3 days (Mw cutoff of 300 kDa). To remove salts from the polymer, the product was further dialyzed against deionized water at room temperature for 2 days and then freeze-dried. The degree of catechol group in resulting production, PEI-DOPA, was determined by an absorbance value at 280 nm using UV-vis spectrophotometer (UV-1601, Shimadzu, Japan), and analyzed by 1H NMR spectroscopy using D2O as a solvent (Bruker DRX 400 spectrometer operating at 400 MHz). Preparation of Magnetite Nanoparticle Clusters (MNCs). MNCs were produced by an oil-in-water (O/W) single emulsion/solvent and evaporation method. First, oleic acid-coated magnetic nanoparticles with an average diameter of 11 nm were synthesized by thermal decomposition of Fe-oleate complex.25 A total of 10 mg oleic acid-coated magnetite nanoparticles dispersed in 1 mL of chloroform was added to 10 mL of deionized water containing 1, 5, 10, or 50 mg of PEIDOPA (the weight ratio of PEI-DOPA to nanoparticles was 0.1:1, 0.5: 1, 1:1, or 5:1, respectively). Sonication was applied to the solution for 5 min using a tip-type Branson sonifier with a duty cycle of 30 and output of 3 in order to form oil-in-water (O/W) emulsion. After solvent

Park et al. evaporation, the remaining polymer and solvent were removed by ultrafiltration using Amicon Ultra-4 centrifugal filter (Mw cutoff of 100 kDa). The purified MNCs were dispersed in fresh deionized water. For comparison, nonclustered magnetite nanoparticles (MNPs) were also prepared by reacting PEI-DOPA and oleic acid-coated magnetic nanoparticles together in the same organic phase. Oleic acid-coated magnetite nanoparticles (11 nm) dispersed in 1 mL of chloroform were mixed with 5 mg of PEI-DOPA dissolved in 1 mL of methanol. After 10 mL of deionized water was added, the mixture solution was emulsified by ultrasonification for 5 min. The residual organic solvent was then eliminated by evaporation. The centrifugation at 2000 rpm for 10 min was repeated twice to purify the magnetite nanoparticles. Preparation of PEG-Functionalized Magnetite Nanoparticle Clusters (PMNCs). For surface coating of PEG on the MNCs, 200 µg of mPEG-SS dissolved in 1 mL of deionized water was added to 3 mL of a stirred solution containing 2 mg of MNCs (molar ratio of mPEG-ss/PEI-DOPA ) 1:14). After reaction for 24 h, the resultant PMNCs were purified using Amicon Ultra-4 centrifugal filter (Mw cutoff of 100 kDa). Similarly, for control experiments, PEG-functionalized MNPs (PMNPs) were also prepared by reacting the same amount of mPEG-ss with MNPs. For rhodamine labeling, 200 µg of PMNCs or PMNPs was reacted with rhodamine B isothiocyanate (2 µg) in 200 µL of deionized water at room temperature overnight. After the reaction, the product was washed with deionized water using Amicon Ultra-4 centrifugal filter (Mw cutoff of 100 kDa). Physical Characterization of Magnetite Nanoparticle Clusters. The hydrodynamic diameter and ζ potential value of the synthesized magnetite nanoparticle clusters were measured by a dynamic light scattering (DLS) instrument (Zetaplus, Brookhaven, NY, U.S.A.) equipped with a He-Ne laser at a wavelength of 632.2 nm. Intensity-weighted particle size distribution and ζ potential values of the particles were measured in 0.1 M PBS solution (pH 7.4) at a physiological temperature (37 °C). The DLS instrument operating with a multimodal particle size distribution pattern was calibrated by using a Nanosphere size standard (Duke scientific Corporation, CA, U.S.A.). The morphology and size were confirmed by transmission electron microscopy (Tecnai F20, Philips). The characterization of clusters was performed via a FT-IR Microscope (IFS66v/s & Hyperion 3000, Bruker Optiks) in the range of 1100-3600 cm-1. The content of PEI within the MNCs was measured using a thermogravimetry analyzer (TG209F3, NETZSCH, Germany) with a heating rate of 5 °C/min under a flow of nitrogen gas. The iron concentration of the samples was quantified by inductively coupled plasma absorption emission spectroscopy (ICPAES; POLY SCAN 61 E, Termo Jarrell Ash, U.S.A.). The magnetization properties of oleic acid-coated magnetite nanoparticles, MNCs, and MNPs were analyzed with a vibrating sample magnetometer (VT800, Riken Denshi Co., Tokyo, Japan). siRNA Binding Ability of PMNCs and PMNPs. The siRNA binding ability of PMNCs and PMNPs were investigated by agarose gel retardation assay. GFP- siRNA was mixed with PMNCs and PMNPs at various weight ratios of Fe3O4 to siRNA (0, 1, 2, 4, 8, and 16) for 15 min. The resultant polyelectrolyte were loaded on 1% agarose gel and then examined by gel electrophoresis at 100 V for 20 min. After staining with ethidium bromide, the gel image was taken under UV illumination. Evaluation of Magnetite Field-Enforced Intracellular Uptake. To visualize the cellular uptake of PMNCs in a time-dependent manner, rhodamine-labeled PMNCs (5 µg/mL) were treated to PC-3 cells seeded in a four-chamber tissue culture slide at a density of 1.5 × 105 cells/well. The cells were treated for 15 min either with or without a permanent magnet (MagnetoFACTOR plate, Chemicell) having a field strength of 70-250 mT and a gradient of 50-130 T m-1. The cells were then incubated for further 15, 30, 60, or 120 min in the absence of the permanent magnet.26 At each time point, the cells were washed with PBS solution and then fixed with 1% (v/v) formaldehyde in PBS solution for 30 min. The fixed cells were monitored by confocal laser scanning microscopy (LSM510, Ziess Pascal, Jene, Germany). To

Magnetite Nanocrystals/PEI for siRNA Delivery quantify the extent of PMNCs internalized by PC-3 cells, the cells were cultivated in a 6-well plate at a density of 3 × 105 cells/well. After cells were treated with PMNCs in the presence or absence of a neodymium magnet for 15 min, they were subsequently incubated for further 2 h without magnetic force. The cells were washed with PBS solution and lysed with 1% (w/v) Triton X-100 in PBS solution. The amount of PMNCs harvested from the cells was quantitatively analyzed using ICP-AES (POLY SCAN 61 E, Termo Jarrell Ash, U.S.A.). Cytotoxicity Assay. PC-3 cells were seeded on a 96-well plate at a density of 1 × 104 cells/well and incubated for 24 h. For cytotoxicity assay, the cells were treated with the culture medium containing branched PEI, PMNPs, or PMNCs at various concentrations of PEI (0-100 µg/mL). After 5 h of incubation, the media in the culture plates were replaced with fresh RPMI media containing 10% (v/v) fetal bovine serum (FBS), and the cells were incubated for an additional 24 h. The cytotoxicity was quantified by the CCK-8 cell viability assay according to the manufacturer’s instruction. To confirm the cytotoxicity in response to magnetofection, PC-3 cells were treated with the polyelectrolyte complexes of GFP siRNA (0.5 µg/mL) prepared with PMNCs and PMNPs at the weight ratio of 24 and 32, respectively. As a control, the polyelectrolyte complexes prepared with branched PEI at the N/P ratio of 16 were also treated. After incubation with or without applying a magnetic field for 15 min, the magnet was removed from the culture plate, and then fresh medium containing 10% (v/v) FBS was added to cells. After 48 h of incubation, cell viability was estimated. Magnetically Triggered Gene Silencing Effect of PMNC/ siRNA Polyelectrolyte Complexes. GFP overexpressing PC-3 cells were cultured in the RPMI medium supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. The cells were seeded in a 12-well plate at a density of 1.5 × 105 cells per well and incubated for 24 h at 37 °C. GFP siRNA (0.5 µg/mL) was mixed with PMNCs or PMNPs at various weight ratios of Fe3O4 to siRNA (8, 16, 24, and 32), and with branched PEI at the N/P ratio of 16. The resultant polyelectrolyte complexes were transfected into PC-3 cells in the absence and presence of a magnetic field for 15 min. The media in the cell culture plate were replaced with fresh media containing 10% (v/v) FBS, and then further incubated for 48 h. The cells were lysed with 1% (w/v) Triton X-100 in PBS solution and centrifuged at 14000 rpm for 5 min to remove cell debris. The amount of GFP in the supernatant was determined by a spectrofluorotometer with an excitation and emission wavelength at 488 and 509 nm, respectively. The relative GFP expression level was calculated as relative percentage against the intensity of GFP expressed in untreated cells. Statistical Analysis. The results were reported as mean ( standard deviation of independent measurements. Statistical analysis was performed with a Student’s t test. Statistical significance was assigned for a P value