Journal of the Korean Physical Society, Vol. 65, No. 10, November 2014, pp. 1658∼1662
Synthesis and Characterization of Photo-functional Magnetic Nanoparticles (Fe3 O4 @HP) for Applications in Photodynamic Cancer Therapy Kyong-Hoon Choi∗ Plasma Bioscience Research Center, Kwangwoon University, Seoul 139-701, Korea
Ki Chang Nam∗ Department of Medical Engineering, Dongguk University College of Medicine, Goyang 410-820, Korea
Ho-Joong Kim Department of Chemistry, Chosun University, Gwangju 501-759, Korea
Jeeeun Min HALLA Energy & Environment, Garak-Dong, Songpa-Gu, Seoul 138-811, Korea
Han Sup Uhm, Eun Ha Choi and Bong Joo Park† Department of Electrical & Biological Physics and Plasma Bioscience Research Center, Kwangwoon University, Seoul 139-701, Korea
Jin-Seung Jung‡ Department of Chemistry, Gangneung-Wonju National University, Gangneung 210-702, Korea (Received 8 December 2013, in final form 12 August 2014) Nowadays, photodynamic therapy (PDT) is a quite promising approach for killing various cancer cells. In this work, we report on photo-functional magnetic nanoparticles conjugated with hematoporphyrin (HP) (Fe3 O4 @HP) via a simple surface modification process. The microstructure and the magnetic properties of the Fe3 O4 nanoparticles were investigated by field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive spectroscopy (EDS), and vibrating sample magnetometry (VSM), and the biocompatibility and the photo-killing activity were evaluated using mammalian cells in vitro to confirm the potential of these particles for use as an agent for PDT application. We have demonstrated that the Fe3 O4 @HP nanoparticles show good biocompatibilities in fibroblast (L-929) and prostate cancer (PC-3) cells and have remarkable photodynamic anticancer activities. Especially, the photo-killing activities for 25, 50, and 100 μg/ml of Fe3 O4 @HP were found to be above 86% (86.6, 99.2, and 99.4%, respectively) in PC-3 cells, demonstrating significantly high anticancer effects on prostate cancer cells, these effects depend on the concentration of the Fe3 O4 @HP nanoparticles. These results indicate that our Fe3 O4 @HP nanoparticles can be useful for PDT, although further studies to evaluate the cell-death mechanisms in vitro and in vivo will be needed to verify the potential for clinical PDT applications. PACS numbers: 07.90.+c Keywords: Photo-functional magnetic nanoparticle, Hematoporphyrin, Biocompatibility, Photodynamic therapy, Prostate cancer cell DOI: 10.3938/jkps.65.1658
∗ These
authors contributed equally to this work.
[email protected] ‡ E-mail:
[email protected] † E-mail:
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Synthesis and Characterization of Photo-functional Magnetic Nanoparticles· · · – Kyong-Hoon Choi et al.
I. INTRODUCTION Fe3 O4 nano- and microspheres have been investigated extensively in the field of materials science during past decade for uses such as ferrofluids, colored pigments, high-density magnetic recording media, chemical sensors, electrophotographic developers and theragnostic materials [1–7]. Especially, they possess unique nanoscale-size-dependent physical and chemical properties that can be controlled in a manner that is not possible in the corresponding bulk materials [8]. The recent research on magnetic particles has led to the development of multifunctional particles by using various surface modification strategies due to their unique magnetic responsiveness, good aqueous dispersion, favorable biocompatibility, and readily-tailored surfaces. With respect to targeting and bioimaging, fluorescein isothiocyanate, folic acid or peptide coated multifunctional magnetic particles have been developed by various groups and their abilities to overcome the limitations of conventional non-specific contrast agents have been demonstrated [9, 10]. Amiri’s group developed a novel theragnostic agent by coating polyethylene glycol fumarate on superparamagnetic colloidal nanocrystal clusters, and that agent has found potential application in magnetic resonance imaging (MRI) and drug delivery [11]. In our previous work, we reported water-soluble photofunctional magnetic nanoparticles (Fe3 O4 @t-PtCP) [12]. Those photofunctional magnetic nanoparticles were utilized as a recyclable photocatalyst to remove the environmental hazard materials in water. In addition, we reported multifunctional magnetic particles conjugated with a photosensitizer and vancomycin as photodynamic inactivation agents for targeting bacteria and capturing or removing them from contaminated sites [13]. However until now, the research on photodynamic cancer therapy using magnetic nanoparticles in the biomedical field has been insufficient. In this article, we report on Fe3 O4 nanoparticles conjugated with hematoporphyrin (HP) (Fe3 O4 @HP) having a photodynamic active function. These Fe3 O4 @HP nanoparticles were designed and synthesized to have an anticancer activity by simple chemical process. The biocompatibility of these particles was confirmed using a method recommended in the document ISO 10993-5 for evaluating the cytotoxicity of biomaterials. Also, the photo-killing activity of Fe3 O4 @HP nanoparticles on PC-3 (human prostate cancer) cells was also evaluated in vitro for using this nanoparticle in clinical applications.
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II. EXPERIMENT 1. Preparation of the Fe3 O4 @HP Nanoparticles
The Fe3 O4 magnetic nanoparticles were fabricated by using a method similar to that in a previous report [14]. Without any other additional chemicals, FeCl3 ·6H2 O (0.54 g) and NaAc (1.5 g) were dissolved in the volume ratio of ethylene glycol / diethylene glycol (VEG /VDEG ) mixture solvent (5:15) to form a clear solution. After 30 min, the as-formed viscous slurry was transferred into a teflon-lined stainless-steel autoclave with an 80 mL capacity. The autoclave was heated to and maintained at 200 ◦ C for 10 h and was cooled naturally to room temperature. The obtained black precipitates were collected after they have been washed with distilled water and absolute alcohol several times and dried at 60 ◦ C for 6 h. In this study, we did not use any other structure-guiding regent to minimize the toxicity of the Fe3 O4 nanoparticles. HP for photo-functionality on the Fe3 O4 nanoparticles was provided by using a wet chemical process similar to the method in our previous report [13]. The precipitated Fe3 O4 nanoparticles (20 mg) were mixed with the solution of HP/tetrahydrofuran (THF) (2.02 × 10−4 M). The mixture solution was agitated for 24 h at room temperature. After the reaction had been completed, the product was washed with THF solution several times.
ticles
2. Characterization of the Fe3 O4 @HP Nanopar-
To determine the size and surface morphology of the Fe3 O4 @HP nanoparticles, we used FE-SEM (Hitachi, SU-70) and TEM (JEOL, JEM-2100F) equipped with an EDS. The crystallographic characteristics and nanostructure of the composite coatings were investigated with an XRD (PANalytical, Pert Pro MPD) using Cu Kα radiation. A VSM (Lakeshore 7300) was utilized to measure the magnetization versus magnetic field loop at room temperature for fields up to kOe. Photoluminescence (PL) and photoluminescence excitation (PLE) spectra were measured on a spectrophotometer (F-4500, Hitachi).
3. Biocompatibility Assessment
To confirm the biocompatibility of the Fe3 O4 @HP nanoparticles, we carried out cytotoxicity tests on L-929 (fibroblast) and PC-3 (prostate cancer) cells as previously described [13,15]. Each pre-cultured cell line was plated in a 24-well plate at 2.0 × 105 cells/mL for L-929 and at 1.0 × 105 cells/ mL for PC-3, and the cells were incubated at 37 ◦ C in 5% CO2 for 24 h. After incubation, the cells were treated with various concentrations
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(0, 6.25, 12.5, 25, 50, 100 g/mL) of Fe3 O4 @HP nanoparticles and were incubated at 37 ◦ C in 5% CO2 for 24 h under dark conditions. The cytotoxicities were evaluated using a cell-counting kit-8 (CCK-8) solution, and the absorbance of each sample was measured at 450 nm with a multi-mode microplate reader (SynergyTM HT, BioTek Instruments, Inc., VT, USA). The relative cell viabilities are presented as a percentage survival in relation to untreated control cells.
4. In-vitro Photo-killing Activity on Prostate Cancer Cell
For the in-vitro photo-killing activity of Fe3 O4 @HP nanoparticles on prostate cancer cell, we used a human prostate cancer cell line, the PC-3 cell line. The cell was used by plating them at 1.0 × 105 cells/mL in a 24-well plate and incubating them at 37 ◦ C in 5% CO2 for 24 h. After that, the cells were incubated with different concentrations (0, 6.25, 12.5, 25, 50, 100 μg/mL) of Fe3 O4 @HP nanoparticles at 37 ◦ C in 5% CO2 for 2 h under dark conditions. The cells were then washed three times with phosphate-buffer solution (PBS), the medium was changed, and the cells were irradiated using a general green-light-emitting diode (LED), with a wavelength in range of λ = 480 ∼ 580 nm and with a maximum wavelength of 515 nm. The LED power was about 20 mW. After irradiation with the LED, the PC-3 cells were incubated for another 24 h, and the following day, the cell viability for determining the photo-killing activity on the cancer cell was measured by using a CCK-8 solution as described above.
III. RESULTS AND DISCUSSION The sizes and the shapes of the Fe3 O4 nanoparticles were investigated by using FE-SEM and TEM. Figure 1(a) shows that the Fe3 O4 nanoparticles were mostly spherical in shape with good size uniformity. The highmagnification TEM images of the Fe3 O4 nanoparticle indicate that a nanoparticle has a rough surface and is composed of small primary grains (Figs. 1(b) and (c)). As shown in the inset of Fig. 1(b), the Fe3 O4 nanoparticle has a single crystalline nature, and the distance between two neighboring planes is approximately 2.98 ˚ A, which is consistent with that between the (220) planes in an inverse spinel-structured magnetite nanoparticle [13]. Figure 1(d) shows the size histogram of the Fe3 O4 nanoparticles, which was obtained by sampling 300 particles in different regions of the FE-SEM images. The histogram indicates that the nanoparticles have good size uniformity and that the average size of a nanoparticle is 93 ± 10.3 nm. The powder XRD pattern of the Fe3 O4 nanoparticles, as shown in Fig. 1(e), provided more detailed structural
Fig. 1. (Color online) Morphology and crystal structure of the Fe3 O4 nanoparticle: (a) FE-SEM image and (b), (c) TEM micrographs of the Fe3 O4 nanoparticle; (d) histogram for the particle size distribution of the Fe3 O4 nanoparticles; (e) XRD pattern of the Fe3 O4 nanoparticles; and (f) EDX spectrum data of the Fe3 O4 nanoparticles.
information. The strong Bragg reflection peaks, marked by their Miller indices ((220), (311), (400), (422), (511), and (440)), are obtained from standard Fe3 O4 powder diffraction data (JCPDS, card 19-0629) [16]. The DebyeScherrer equation (Dhkl = kλ/β cos θ) was used to estimate an average crystallite size from the XRD patterns (fitted by using a convolution of Lorentzian functions). The average primary grain size of a Fe3 O4 nanoparticle is about 8.5 nm. The EDS analysis of the Fe3 O4 @HP nanoparticles illuminated by an electron beam revealed the existence of Fe, C, O, and Cu elements (Fig. 1(f)). The Fe:O molar ratio is 3.0 : 4.4, similar to that of the stoichiometric composition. The Cu peaks in the EDS spectrum are thought to result from the carbon-coated copper TEM grid. The room-temperature hysteresis loops of the pure Fe3 O4 and the Fe3 O4 @HP nanoparticles were measured using a VSM. As shown in Fig. 2, the magnetization curves of both nanoparticles exhibit no hysteresis, and no coercivity is reached, not even at the highest magnetic field. This indicates that these magnetic particles have a superparamagnetic behavior. The pure Fe3 O4 nanoparticles show a high saturation magnetization value of 60.1 emu/g whereas the high saturation value of the surfacemodified Fe3 O4 @HP nanoparticles is 38.6 emu/g. The difference in the saturation values is attributed to the diamagnetic contribution of the HP molecules. Figure 3 shows the photoluminescence (PL) and the photoluminescence excitation (PLE) spectra of the pure HP and the Fe3 O4 @HP nanoparticles in THF. The peak at 400 nm is the Soret band of HP, and the Q bands are located at 500, 532 and 568 nm. At the excitation wavelength of 400 nm, the pure HP produces two strong emission peaks located at 625 nm and 692 nm, and the Fe3 O4 @HP nanoparticles provide very similar peaks. Singlet oxygen generation from the Fe3 O4 @HP
Synthesis and Characterization of Photo-functional Magnetic Nanoparticles· · · – Kyong-Hoon Choi et al.
Fig. 2. (Color online) Room-temperature magnetic hysteresis loops of pure Fe3 O4 nanoparticles and Fe3 O4 @HP nanoparticles.
Fig. 3. (Color online) PL and PLE spectra of pure HP and Fe3 O4 @HP nanoparticles in THF. Excitation and detection wavelengths are 400 nm and 625 nm for the PL and PLE spectra, respectively.
nanoparticles is also confirmed with indirect detection of 1,3-diphenyl-isobenzofuran (DPBF) photodegradation to confirm the photodynamic activity of the Fe3 O4 @HP nanoparticles (Data not shown). To confirm the biocompatibility of the Fe3 O4 @HP nanoparticles, we evaluated the cytotoxicity of the Fe3 O4 @HP nanoparticle itself with different concentrations on fibroblast (L-929 cells) and prostate cancer (PC3 cells) cells under dark condition, by using the recommended method (International standard ISO 10993-5) [15]. This is a very important step to evaluate the potentials of the Fe3 O4 @HP nanoparticles for clinical applications. As shown in Figs. 4(a) and (b), the Fe3 O4 @HP nanoparticles did not show any cytotoxicity on L-929 and PC-3 cells in the concentration range of 0 to 100 μg/ml.
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Fig. 4. (Color online) Cytotoxicities of Fe3 O4 @HP on (a) L-929 cells and (b) PC-3 cells. Cells were cultured with different concentrations of Fe3 O4 @HP for 24 h at 37 ◦ C under dark conditions. (c) Photo-killing activity for each concentration of Fe3 O4 @HP in PC-3 cells. (d) Fluorescence images for viable PC-3 cells. Cells were incubated with different concentrations of Fe3 O4 @HP for 2 h in the dark prior to LED irradiation for 30 min. After irradiation, the cells were then incubated for another 24 h, and the cell viabilities were measured by using CCK-8 solution. Data are expressed as a mean ± standard deviation (n = 6) and were analyzed by using the Student’s t-tests. Statistical significance was considered as p < 0.05 (∗ p < 0.05, ∗∗ p < 0.005 vs. control at the same time).
The cell viabilities of the two kinds of cells were more than 91%. These results indicate that the Fe3 O4 @HP nanoparticles have good biocompatibility on L-929 and prostate cancer (PC-3 cells) cells and can be safely used for clinical photodynamic cancer therapy. For evaluating the photo-killing activity of the Fe3 O4 @HP nanoparticles, we selected and used prostate cancer (PC-3 cells) cells, which are metastatic prostatecancer cells. Because prostate-cancer is the most frequently-diagnosed cancer in men and the third cause of cancer mortality in the United States [17,18] a need exists to develop a novel and effective therapy for treating metastatic prostate-cancer. The PC-3 cells incubated with different concentrations of Fe3 O4 @HP nanoparticles were irradiated using a LED for 30 min and then incubated another 24 h in the dark. The degrees of photokilling activity were quantified by using CCK-8 solution as shown in Fig. 4(c), and images of live cells were taken under a fluorescence microscope after the cells had been stained with a fluorescence diacetate (FDA) dye, which is a cell-permeant esterase substrate that can serve as a viability probe, as shown in Fig. 4(d). Figure 4(c) shows the PC-3 cell viability normalized to the control. The cell viabilities were 95.7 ± 2.2, 72.8 ± 2.2 (p < 0.036), 13.2 ± 2.2 (p < 0.004), 0.8 ± 2.2 (p < 0.0015), and 0.6 ± 2.2 (p < 0.0016)% in 6.25, 12.5, 25, 50, and 100 μg/ml of Fe3 O4 @HP nanoparticles, respectively. Es-
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Journal of the Korean Physical Society, Vol. 65, No. 10, November 2014
pecially, the photo-killing activities in 25, 50, and 100 μg/ml of Fe3 O4 @HP nanoparticles were 86.6, 99.2, and 99.4%, respectively, demonstrating significantly-high anticancer effects that depended on the concentration of Fe3 O4 @HP nanoparticles in the prostate-cancer cell. Figure 4(d) shows fluorescence images of live cells stained with FDA dye 24 h after irradiation. The FDA dye passes through a living cell’s membrane, accumulates inside the cell, and exhibits green fluorescence when excited at 488 nm, as shown in Fig. 4(d), therefore, FDA can be used for detecting the cell’s viability [19]. The images also represent the dose-dependent photokilling effects in PC-3 cell, as in Fig. 4(c). These results demonstrate that the Fe3 O4 @HP nanoparticles internalized into the PC-3 cells can generate high levels of singlet oxygen, which is a key mediator of cell death during irradiation, leading to high level of photo-killing activity in PC-3 cells, as shown in Figs. 4(c) and (d).
IV. CONCLUSION In this study, we successfully fabricated biocompatible photo-functional magnetic nanoparticles conjugated with HP (Fe3 O4 @HP) via a simple surface modification process for PDT applications and we confirmed a remarkable concentration-dependent photodynamic anticancer activity of the Fe3 O4 @HP nanoparticles in human prostate cancer cell when using LED irradiation. These results indicate that the Fe3 O4 @HP nanoparticles have potential as a therapeutic agent for PDT, although further studies to evaluate the cell death mechanisms in vitro and in vivo will be needed to verify the potential for clinical photodynamic anticancer applications.
ACKNOWLEDGMENTS This work was supported financially by the National Research Foundation of Korea (NRF, #2010-0029418). J.-S. Jung thanks the Research Institute of Natural Science of Gangneung-Wonju National University.
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