Intracellular translocation of superparamagnetic iron oxide nanoparticles encapsulated with peptide-conjugated poly(D,L lactide-co-glycolide)

JOURNAL OF APPLIED PHYSICS 97, 10Q913 共2005兲

Intracellular translocation of superparamagnetic iron oxide nanoparticles encapsulated with peptide-conjugated poly„D,L lactide-co-glycolide… Seung-Jun Lee Department of Chemical & Biomolecular Engineering, KAIST, Daejeon, Republic of Korea

Jong-Ryul Jeong and Sung-Chul Shin Department of Physics, KAIST, Daejeon, Republic of Korea

Yong-Min Huh, Ho-Taek Song, and Jin-Suck Suh Diagnostic Radiology, Yonsei University Medical Center, Seoul, Republic of Korea

Young-Hwan Chang Division of Natural Science, KAIST, Daejeon, Republic of Korea

Bong-Sik Jeon and Jong-Duk Kim Department of Chemical & Biomolecular Engeering, KAIST, Daejeon, Republic of Korea

共Presented on 9 November 2004; published online 17 May 2005兲 In this study, we propose the use of iron oxide nanoparticle encapsulated with peptide-conjugated poly共D,L lactide-co-glycolide兲 共PLGA兲 as a potent intracellular carrier for diagnosis agent. The iron oxide 共␥-Fe2O3兲 nanoparticles were prepared by a chemical coprecipitation method of ferric and ferrous ions in an alkali solution. Arg peptide 共RRRRRRRRCK-FITC兲 were conjugated to the PLGA via a simple coupling reaction between maleimide-derivatized PLGA and thiol-terminated Arg peptide. The ␥-Fe2O3-PLGA-Arg-FITC nanoparticle was then prepared by an emulsification-diffusion technique. A confocal laser scanning microscopy revealed that the ␥ -Fe2O3-PLGA-Arg-FITC nanoparticle was effectively adsorbed onto the membrane of stem cells and delivered into the nuclei without cytotoxicity. Magnetic properties of the ␥-Fe2O3-PLGA-Arg-FITC nanoparticle were observed by measuring the zero-field-cooled/ field-cooled magnetization and magnetic hysteresis loop using a superconducting quantum interference device magnetometer from 5 K to 300 K. © 2005 American Institute of Physics. 关DOI: 10.1063/1.1853933兴 I. INTRODUCTION

peptides-conjugated magnetic nanoparticles as a potent intracellular carrier for MR molecular image diagnosis agent.

In recent years, superparamagnetic nanoparticles have been evaluated as magnetic resonance 共MR兲 molecular imaging contrast agent, especially for liver, spleen, and breast. MR imaging is a noninvasive technique routinely used clinically for diagnostic imaging. However, intrinsic sensitivity of MR technique is significantly low in comparison with conventional optical and nuclear imaging. Therefore, to improve the sensitivity of MR up to the level where detection of molecular markers becomes possible, special contrast agents amplifying the MR signals need to be designed. Significant signal amplification can be achieved if the contrast agent is allowed to accumulate in the target cells by passive endocytosis, or by an active transporter system such as a transferrin receptor that shuttles targeted contrast agent into the cell. Therefore, the development of superparamagnetic iron oxide nanoparticles and iron oxide nanoparticles encapsulated have been intensively pursued because of their technological and fundamental scientific importance.1,2 Superparamagnetic nanoparticles offer a high potential for several applications in different areas such as ferrofluids, color imaging, magnetic refrigeration, detoxification of biological fluids, magnetically controlled transport of anticancer drugs, magnetic resonance imaging contrast enhancement and magnetic cell separation.3,4 In this study, we propose here the use of 0021-8979/2005/97共10兲/10Q913/3/$22.50

II. EXPERIMENT

Superparamagnetic nanoparticles of maghemite 共␥Fe2O3兲 were prepared by a chemical coprecipitation method of ferric and ferrous ions in alkali solution. The detailed procedure was described in a previous report.4 The reaction steps in our process are as follows: FeCl2共1 mol兲 + FeCl3共2 mol兲 → Fe3O4 → ␥-Fe2O3 A molar ratio of Fe共II兲 / Fe共III兲 = 0.5 was dissolved in water with sonication. The resulting solution was poured with piezoelectric nozzle 共nozzle size: 50 ␮m, 5 ⫻ 10−7 ml/ drop兲 method into an alkali solution. The x-ray diffraction 共XRD兲 pattern matches well with that of ␥-Fe2O3 共powder diffraction file, JCPOS card no. 25-1402兲. All organic solvents used in this experiments were either high performance liquid chromatography 共HPLC兲 grade or american chemical society analytical grade reagents. In Fig. 1共a兲, we demonstrate procedure for preparing the iron oxide 共␥-Fe2O3兲 nanoparticles encapsulated with Arginine 共Arg兲 peptide 共RRRRRRRRCK-FITC兲 conjugated with poly 共D,L lactide-co-glycolide兲 共PLGA兲. It should be noted that Arg peptide is well known as a cell penetration peptide. Cell penetration peptide means peptides leaded to intracellu-

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FIG. 2. Particle size distributions of the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles measured by DLS.

FIG. 1. 共a兲 The schematic diagram ␥-Fe2O3-PLGA-Arg-FITC nanoparticles. 共b兲 arginine-peptide.

for preparing The sequence

the of

lar translocation across the cellular membrane into the cytoplasm and nucleus by a seemingly energy-independent mechanism.5,6 Figure 1共b兲 shows the sequence of Arg peptide. The fluorescein 共FITC兲 conjugated Arg peptide was prepared by Peptron 共Daejeon, Korea兲. The FITC was conjugated to observe the intracellular translocation of magnetic nanoparticles into the cell. The Arg peptide was synthesized by solid phase peptide synthesis and purified 共⬎95% 兲 by using reverse-phase HPLC, and their molecular masses were determined by mass spectroscopy. The intracellular translocation was investigated using the human mesenchymal stem cells 共MSC兲, obtained from Yonsei medical center. The cellular internalization into MSC was examined by using a confocal laser scanning microscope 共CLSM兲. XRD measurement using Cu K␣ radiation was performed to identify the crystal structure of iron oxide nanoparticles. Magnetization measurements were performed using a superconducting quantum interference device 共SQUID兲 magnetometer from 5 K to 300 K. III. RESULTS AND DISCUSSION

To encapsulate the magnetic nanoparticle using peptideconjugated PLGA, we first combined FITC conjugated Arg peptide with PLGA. The Arg peptide was conjugated to PLGA via a simple coupling reaction between maleimidederivatized PLGA and thiol-terminated Arg peptide. Carboxylic acid end group of PLGA was activated to the succinimidyl ester using N-hydroxysuccinimide and 1,3dicyclohexylcarbodiimide, and then, was converted to primary amine groups using an excess amount of hexamethylene diamine. Maleimide-terminated PLGA was then prepared by reacting N-succinimidyl 4-共4-maleeimidophenyl兲 butyrate to the primary amine terminated PLGA. Finally, PLGA was conjugated to the Arg peptide by the coupling reaction between maleimide group of PLGA and the sulfhy-

dryl group of the Arg peptide. The resulting Arg peptide conjugated PLGA was purified by dialysis against excess de-ionized distilled water and freeze drying. After preparing the Arg-peptide conjugated PLGA, the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles were prepared by using an emulsification-diffusion method.2 First, 200 mg of peptide-conjugated PLGA was dissolved in 10 ml of ethylacetate 共EtAc, Fluka兲 forming 2 wt % of polymer solution, and then 60 mg of magnetic nanoparticle was added. The mixture was emulsified for 5 min with a bath-type sonicator. The organic mixture with magnetic nanoparticle was added into 20 ml of an aqueous phase containing stabilizer. After mutual saturation of organic and continuous phases, the mixture was emulsified for 7 min with a high speed homogenizer 共about 20 000 rpm兲, and then water is added to o / w emulsion solution under sonicator. The subsequent addition of water dilutes the solvent concentration and it causes extraction of solvent from the polymer solution, which leads to the nanoprecipitation of polymer. The conjugation degree of the Arg peptides at the surface of ␥-Fe2O3 was about 18.7% in EtAc, as determined by a UV absorbance 关UV absorbance = ␧ ⫻ concentration共M兲 ⫻ length of the UV cell 共cm兲, ␧ = 80 000M −1 cm−1兴 where ␧ is molecular extinction coefficient of the FITC.7 Figure 2 shows the size distribution of ␥-Fe2O3-PLGA-Arg-FITC nanoparticles measured by dynamic light scattering 共DLS兲. As seen in Fig. 2, it shows very narrow size distribution from 111 nm to 116 nm, which is very suitable for intercellular application. To investigate the magnetic properties of the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles, 共field-cooled/zerofield-cooled兲 共FC/ZFC兲 measurements have been made using a SQUID magnetometer. The SQUID measurements revealed superparamagnetic nature of the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles. Figure 3 shows the FC and ZFC curves for the 115 nm ␥-Fe2O3-PLGA-Arg-FITC nanoparticles from 5 K to 300 K at 200 Oe. The ZFC and FC curves coincide above 180 K and separate below 180 K. The ZFC curve shows a broad peak at Tmax ⬃ 102± 5 K indicative of a characteristic blocking temperature for superparamagnetic particles.8–11 To investigate the intracellular translocation of the ␥ -Fe2O3-PLGA-Arg-FITC nanoparticles, the MSC were cultured in Dulbecco’s modified eagle medium 共DMEM, Gibco

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FIG. 3. The FC/ZFC curve for the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles.

BRL, Gaitthersburg, USA兲 supplemented with 1 % 共v / v兲 antibiotics and 10% 共v / v兲 fetal bovine serum. The cells grown in 35 mm ⌬T culture dishes were incubated with 1.0– 25 mg/ ml of ␥-Fe2O3-PLGA-Arg-FITC nanoparticles at 37 ° C in a 5% 共v / v兲 CO2 humidified incubator for 2 – 18 h. The medium was then removed and the cells were washed three times with fresh medium. The cellular internalization of the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles into the MSC was examined by using a CLSM. Figure 4共a兲 shows the fluorescence images of stem cells incubated with the ␥ -Fe2O3-PLGA-Arg-FITC nanoparticles at 200 ␮g / ml of iron oxide nanoparticles concentration for 2 h. Figure 4共a兲 clearly

shows that the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles were effectively adsorbed onto the membrane of stem cells and delivered into the nuclei. To verify this phenomena, the iron oxide nanoparticles were stained with prussian blue dye called Fe dye, ferric ferrocyanide, Fe4关Fe共CN兲6兴3, at about 200 ␮g / ml of ␥-Fe2O3-PLGA-Arg-FITC nanoparticles concentration. As shown in Fig. 4共b兲, the optical microscope image for prussian blue staining Fe elements clearly represent that the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles have superior intracellular translocation ability into the cell. The intracellular blue color in Fig. 4共b兲 shows the iron oxide nanoparticles which are transferred into the stem cell, i.e., most of iron oxide nanoparticles effectively accumulated, adsorbed onto the membrane of stem cells, and delivered into the nuclei in the cell. Cytotoxicity of intracellular carriers is one of the most important parameters to be checked. To deal with this concern, cytotoxicity was checked using a 3,4,5dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide 共MTT, Sigma兲 assay and a lactate dehydrogenase 共CytoTox 96 kit, Promega, Madison, USA兲 release assay. In this test, two-day cell incubation with peptide-conjugated ␥-Fe2O3-PLGA-Arg did not show significant cytotoxicity up to 200 ␮g / ml of iron oxide nanoparticle concentration. IV. CONCLUSIONS

The ␥-Fe2O3-PLGA-Arg-FITC nanoparticles were prepared by a simple coupling reaction between the PLGA and thiol-terminated Arg peptide. The SQUID measurements revealed superparamagnetic nature of the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles. We witness that the ␥-Fe2O3-PLGA-Arg-FITC nanoparticle was effectively adsorbed onto the membrane of stem cells and delivered into the nuclei without cytotoxicity. ACKNOWLEDGMENTS

This research was partially supported by Center for Ultramicrochemical Process Systems project and by Ministry of Science & Technology project through the Creative Research Initiatives Project. 1

FIG. 4. 共a兲 CLSM image of stem cells after incubation with the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles at 37 ° C for 2 h. 共b兲 The optical microscope image for prussian blue staining with the ␥-Fe2O3-PLGA-Arg-FITC nanoparticles.

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