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Magnetic manipulation of bacterial magnetic nanoparticle-loaded neurospheres† Jaeha Shin,a Kyung-Mee Lee,b Jae Hyup Lee,b Junghoon Lee*ac and Misun Cha*c Specific targeting of cells to sites of tissue damage and delivery of high numbers of transplanted cells to lesion tissue in vivo are critical parameters for the success of cell-based therapies. Here, we report a promising in vitro model system for studying the homing of transplanted cells, which may eventually be applicable for targeted regeneration of damaged neurons in spinal cord injury. In this model system, neurospheres derived from human neuroblastoma SH-SY5Y cells labeled with bacterial magnetic
Received 30th September 2013, Accepted 21st February 2014
nanoparticles were guided by a magnetic field and successfully accumulated near the focus site of
DOI: 10.1039/c3ib40195b
bacterial magnetic nanoparticles to develop successful stem cell targeting strategies during fluid flow,
the magnetic field. Our results demonstrate the effectiveness of using an in vitro model for testing which may ultimately be translated into in vivo targeted delivery of cells through circulation in various
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tissue-repair models.
Insight, innovation, integration We present a promising in vitro model system to study the homing of transplanted stem cells, which may eventually be applicable for targeted regeneration of damaged neurons in spinal cord injury. In this model system, neurospheres were labeled with bacterial magnetic nanoparticles (BMPs) to enhance the homing of transplanted cells to a target site. BMP-loaded neurospheres were guided by a magnetic field and successfully accumulated near the focus site of the magnetic field. This study shows the effectiveness of using an in vitro model for testing BMPs to develop successful stem cell targeting strategies during fluid flow, which may ultimately be translated into in vivo targeted delivery of cells through the circulation in various tissue-repair models.
Introduction The clinical application of stem cells has shown promise for cell transplantation therapies;1–6 however, identifying the optimum delivery strategy of the cells to the injury site remains a major challenge. Targeting and differentiation of stem cells at sites of injury and repair are exciting areas of research in regenerative medicine. Delivery of stem cells to specific targets in vivo has been achieved via direct needle injection to the site of delivery,3,6–9 local infusion,3,10,11 or preferential homing or distribution following systemic administration. However, these
a
School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-744, South Korea b Department of Orthopedic Surgery, College of Medicine, Seoul National University, SMG-SNU Boramae Medical Center, 39 Boramae Gil, Dongjak-Gu, Seoul, 156-707, South Korea c Inter-University Semiconductor Research Center, Seoul National University, Seoul, 151-744, South Korea. E-mail: [email protected], [email protected]; Fax: +82-2-877-9104; Tel: +82-2-880-9101 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3ib40195b
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delivery methods generally suffer from a lack of precise localization, low repeatability, and local damage to the injection site. The versatile intrinsic properties of magnetic particles render them useful for targeted cell therapy, where magnetic carriers loaded with cells can be directed or guided by means of a magnetic field gradient toward biological targets.12,13 Recent studies have shown that magnetic labeling of cells is relatively easy and safe.14–18 However, only a few studies have used magnetic fields to guide magnetic particle-loaded cells to specific targets because of low uptake at the site of injury.19,20 Herein, we present a promising model system that uses magnetic particle loading of cell spheres to enhance the homing of transplanted cells to a target site; this system may therefore be useful for facilitating the regeneration of damaged neurons in spinal cord injury. The bacterial magnetic nanoparticles (BMPs) employed in our study are inherently biocompatible and disperse well in aqueous solutions because of a stable lipid membrane wrapping the magnetic core.21–23 This lipid membrane contains lipids and proteins, such as phosphatidylserine, phosphatidylethanolamine, glycolipids, and sulfolipids.21–23 In spite of concerns about embedded proteins of lipid membrane, the BMPs showed no significant toxicity in vitro and in vivo.24–28
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BMP-loaded neurospheres (BMP-NSs) of SH-SY5Y cells, a cell line commonly used for in vitro neurological studies,29 were investigated with regard to targeted delivery and transplantation into the lesion site in an in vitro model of the spinal cord. Cells delivered in the spheres demonstrated improved delivery efficiency relative to individual cells delivered in suspension.
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Materials and methods BMP preparation BMPs were extracted from the magnetotactic bacterium (MTB) Magnetospirillum sp. AMB-1 (ATCCs 700264), which was cultured in magnetic spirillum growth medium (MSGM) for 5 d in a shaking incubator at 30 1C under anaerobic conditions.30 After sufficient culture, the cells were centrifuged at 5000 rpm for 25 min and were then lysed using a homogenizer (VCX500, Sonics & Materials, USA) for 30 min. BMPs were collected using a neodymium iron boron (NdFeB) magnet and washed five times with 1 PBS. The BMPs collected were dispersed in 1 PBS and sterilized by autoclaving (121 1C, 15 min). The concentration of the extracted BMPs was determined by inductive coupled plasma-atomic emission spectrometry (ICP-AES; ICPS-7500, Shimadzu, Japan).
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cacodylate buffer for 2 h at 4 1C. The cells were then briefly washed twice with distilled water, stained with 0.5% uranyl acetate for 30 min at 4 1C, and dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, and 100%) for 10 min each. The samples were treated with 100% propylene oxide twice for 10 min and embedded in Spurr’s resin. Ultrathin sections were prepared using an ultramicrotome (MT-X, RMC, USA) and mounted on copper grids. The sections were examined using a TEM (JEM-1010, JEOL, Japan). BMPs were labeled with fluorescein isothiocyanate (FITC) to allow tracking of the location of internalized BMPs in the cells using confocal laser scanning microscopy. After delivery of FITC-labeled BMPs, the cells were fixed with 4% paraformaldehyde in 1 PBS for 15 min at room temperature. After two washes with wash buffer, i.e., 0.1% Triton X-100 in 1 PBS, the fixed cells were permeabilized and blocked using 1% BSA and 0.1 Triton X-100 in 1 PBS for 20 min. Actin filaments were then stained with TRITC-conjugated phalloidin (1 : 200) for 50 min at room temperature. The cells were briefly washed three times with 1 PBS, and the DNA in the nuclei was counterstained using dilute DAPI in 1 PBS (1 : 1000) applied three times for 5 min each. The stained cells were examined using a confocal laser scanning microscope (LSM710, Carl Zeiss, Germany).
Cell culture and intracellular uptake of BMPs SH-SY5Y cells, a human-derived neuroblastoma cell line, were cultured in Dulbecco’s modified Eagle medium (DMEM; Thermo Scientific, USA) containing 10% (v/v) fetal bovine serum (FBS; Thermo Scientific, USA) and 1% penicillin/streptomycin (Thermo Scientific, USA) at 37 1C in a humidified atmosphere containing 5% CO2. The cells were cultured to 80% confluency in BMP-free culture medium and were then treated with BMPs (10 mg ml1 applied to more than 106 cells) in serum-reduced medium (1% FBS) and incubated overnight to allow internalization of BMPs. The BMP-loaded cells (BMP-SH) were separated from non-BMPloaded cells using a magnet. For neurosphere formation, the cells were cultured in a floating culture for 5 d. Cell staining The cell membrane was labeled with red fluorescent dye (PKH26, Sigma-Aldrich, USA) for tracking of single cells and neurospheres in the target site; individual cells were cultured under appropriate conditions for subsequent experiments and neurospheres were formed. Stained cells were examined using a fluorescence microscope (Nikon, Japan). Microscopy Transmission electron microscope (TEM) imaging of the cells was performed after overnight treatment with the BMPs to confirm internalization of the BMPs. The treated cells were immersed in modified Karnovsky’s fixative solution (2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate buffer) for 4 h at 4 1C. After three washes for 10 min in 0.05 M sodium cacodylate buffer, the cells were post-fixed with 1% osmium tetroxide in 0.05 M sodium
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Exposure of static magnetic field Static magnetic fields (SMFs) were generated with a neodymium iron boron (NdFeB) magnet (50 25 25 mm) with an average flux density of 480 10 mT, as measured by a Gauss meter (TM-701, Kanetec, Japan). Magnets were placed under the plastic culture dishes when changes in cell growth were evaluated. Because the SMF intensity decreases according to the square of the distance from the magnet, the magnetic force on the cells was calculated taking the thickness of the bottom of the culture dish (1.0 mm) into account. The SMF was applied continuously at the bottom of the culture dish for up to 6 d. When neurite outgrowth was measured, the magnets were placed laterally on both sides of the culture dish (Fig. S2, ESI†), and the cells were treated with retinoic acid (RA, all trans-retinoic acid; Sigma, USA) for 1 week. MTS assay The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed to evaluate the cytotoxicity of the BMPs and viability of the cells. The cells (5 103 cells per well) were seeded on a 96-well plate with 120 ml of culture medium. After the cells were sufficiently cultured, 25 ml of CellTiter 96s AQueous One Solution Reagent (Promega, USA) was added into each well. The plate was then incubated at 37 1C for 1–4 h in a humidified 5% CO2 atmosphere, and the absorbance was recorded at 490 nm using a microplate reader (Sunrise, Tecan, Switzerland). All the assays were performed in three separate sets of experiments, and the absorbance of control cells was defined as 100% viability in every assay.
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Measurement of neurite outgrowth Differentiation of SH-SY5Y and BMP-SH cells was induced by treating them with 10 mM retinoic acid (RA) in serum-reduced medium; the medium was changed every 3 d. Neurite outgrowth of the cells was assessed after 7 d of differentiation with or without exposure to SMF. Neurite length was measured and classified into three levels according to the length, i.e., 0–50 mm, 50–100 mm, or over 100 mm. In total, 200 cells in each condition were analyzed in three separate sets of the experiments, with the standard error of the mean being recorded. Manipulation of BMP-loaded cells and neurospheres BMP-loaded individual SH-SY5Y cells (BMP-SHs) and BMP-NSs were prepared as described above and labeled with a red fluorescent dye to track the movement. In the non-flow condition, the magnetic field was produced using an NdFeB magnet and the motion of the BMP-NSs was observed within 2 mm from the magnet, where the field gradient was approximately 40 T m1. To study the delivery of BMP-SHs and BMP-NSs into a simulated spinal cord under flow conditions, a glass capillary tube with a diameter of 1 mm was employed. The inner surface of the tube was coated with fibronectin to permit attachment of the cells on the surface after delivery. The sterilized capillary tube was filled with a solution of 100 mg ml1 fibronectin in 1 PBS, incubated overnight at room temperature, and then dried. A syringe pump (KD Scientific, USA) was used to introduce the cells dispersed in culture medium (104 cells or spheres) into the capillary tube with a stable flow rate of 22.6 ml h1. As the cells flowed in the tube, a focused magnetic field was applied to a target point to capture the cells using an edge of the magnet that generated approximately 100 T m1 of field gradient. The capillary tube filled with the culture medium and the captured cells were incubated at 37 1C for 20 h in a humidified 5% CO2 atmosphere after delivery of the cells. The movement of the BMP-SHs and BMP-NSs under flow conditions and attachment of the cells on the inner surface of the tube were examined using a fluorescence microscope (Nikon, Japan).
Results and discussion
Fig. 1 Observation of BMPs internalized SH-SY5Y cells using (a) confocal microscopy; (b) TEM of the cell, scale bars: 10 mm, red: actin filaments, blue: nucleus, green: BMPs; (c) cytotoxicity of BMPs to SH-SY5Y cells with various concentrations with standard error of the mean (SEM) and p-value from unpaired t-test.
Uptake and cytotoxicity of BMPs Magnetospirillum sp. AMB-1 is an MTB that is widely used because it grows well under laboratory conditions. BMPs from AMB-1 contain a well-crystallized magnetite (Fe3O4) core and an enveloping magnetosome membrane that has a lipid bilayer and embedded proteins.21–23 These BMPs are highly crystallized, with few defects, a uniform size, and ferrimagnetic properties that are difficult to achieve in synthesized magnetic nanoparticles at room temperature.21–23 The uptake of BMPs by SH-SY5Y cells was directly confirmed by electron microscopy. The BMPs were labeled with FITC to track the location of internalized BMPs by using confocal laser scanning microscopy. As shown in Fig. 1a, the BMPs were internalized well into the SH-SY5Y cells, and most of the internalized BMPs were found in the cytosol. The TEM images
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clearly showed that the internalized BMPs were mostly located in the endosome or lysosome in the cytosol; a few BMPs escaped from the endosome were found in the cytosol after overnight uptake (Fig. 1b, right upper and lower boxes). Endocytosis is a well-known mechanism for cellular uptake of nanoparticles, and most of the internalized particles are typically localized in the endosome or lysosome.31,32 The uptake efficiency of the BMPs in SH-SY5Y cells was 43.42% when BMPs at a concentration of 20 mg ml1 were delivered to 106 cells (Fig. S1, ESI†); this value is comparable with the uptake of BMPs by HeLa cells,33 as evaluated by measuring the ferric concentration using ICP-AES (ICPS-7500, Shimadzu, Japan). BMPs showed no cytotoxicity against SH-SY5Y cells up to a concentration of 300 mg ml1 for 5000 cells
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during culture for 3 d. The viability of the cells decreased at BMP concentrations over 75 mg ml1 after 6 d of incubation; therefore, SH-SY5Y cells were treated with BMP concentrations lower than 75 mg ml1 in subsequent studies (Fig. 1c).
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Enhancement of cell growth and neuronal differentiation with internalization of BMPs In a previous study,33 we reported the in vitro biological effects of the communications between internalized BMPs and an external SMF. Combination of the BMPs and SMF induced modulation of GPCRs-mediated signal transduction, thereby alteration of cell structure and the enhanced cell growth were caused. Also, an antiapoptotic effect was induced by the synergetic interaction of magnetism. In this study, the magnetic response of BMP-SH cells was investigated under continuous exposure to an SMF. The SMF (480 10 mT) was constantly applied to investigate cell growth at the bottom of the dish for up to 6 d. However, the synergetic effect of the BMPs and SMF was not observed in SH-SY5Y cells. Internalization of BMPs induced a substantial increase in SHSY5Y cell growth (up to 30% in 6 days), whereas the effect of SMF exposure was negligible regardless of BMP internalization (Fig. 2a). MFs are thought to influence the viability of cells because of the diamagnetic properties of the molecules in the cell membrane.34 Some types of cells, however, have been reported to be essentially unaffected by exposure to MFs,35,36 such as the SH-SY5Y cells. Neuronal differentiation of the BMP-SH cells was induced by constant treatment with RA (10 mM ml1) for 7 d under SMF conditions. The SMF (100–220 mT) was applied to cells laterally as neuronal differentiation was induced by RA treatment (Fig. S2 and S3, ESI†). The neurite length was measured and classified according to three levels of length, i.e., 0–50 mm, 50–100 mm, or over 100 mm, after 7 d of differentiation (Fig. 2b and c). The length of the neurites was significantly higher in BMP-SH cells (regardless of their exposure to SMF) than in BMP-free cells, particularly with regard to the ratio of neurites longer than 50 mm (Fig. 2b and c). Neurite outgrowth is a major morphological alteration in neuronal differentiation induced by RA.37 Improvement of neurite outgrowth using magnetic nanoparticles has been previously reported in another neural cell line, PC12 cells.38 It has been proposed that magnetic nanoparticles could be exploited to enhance or accelerate nerve regeneration and to provide guidance for regenerating axons.38 Nanoparticles may also be used to create mechanical tension to stimulate the growth and elongation of axons. Our results imply that targeted magnetic cell delivery to injury sites without side effects may be possible using SMF and that BMP-SH cells delivered to lesion sites magnetically may afford efficient regeneration and healing. BMP-loaded neurosphere culture A neurosphere is a free-floating spherical cluster of neural stem cells obtained from floating cultures in vitro.39 Neurospherederived cells have been successfully used in models of
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Fig. 2 (a) Cell growth of SH-SY5Y cells under an SMF with SEM and pvalue from unpaired t-test (**p o 0.005, ***p o 0.0001), (b) measurement of neurite outgrowth length and (c) microscope images of SH-SY5Y cells with treatment of RA (10 mM) under an SMF, scale bars: 50 mm.
neurological injury such as experimental autoimmune encephalomyelitis and spinal cord injury.40–42 Therefore, neurospheres may be useful for the delivery of therapeutic cells to lesion sites. SH-SY5Y human neuroblastoma cells can grow in neurospheres under floating culture conditions without requiring special treatment such as serum and growth factors. We confirmed that the BMP-SH cells formed neurospheres with
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after 5–10 d of free-floating culture. Also, as shown in Fig. 3b, the green spots indicating FITC-labeled BMPs were maintained in the cells as they grew into neurospheres. The neurospheres formed from single BMP-SH cells theoretically have the same amount of BMPs found in individual BMP-SH cells. Moderately grown neurospheres (diameter, B100 to 200 mm) were isolated and cultured on an extracellular matrix (ECM)coated surface to investigate transplantation of neurospheres after delivery to the target site. The neurospheres attached to the surface, and individual cells from the neurospheres migrated to the surrounding area (Fig. 3c) in similar patterns as control cells (Fig. S4, ESI†). The results show that the BMP-NSs could be applied for delivery of therapeutic cells with similar functionality of neurospheres from control cells. Theory of manipulation of BMP-NSs The BMP-NS has the potential to be spatially manipulated by virtue of its free-floating form and the magnetic reactivity of the BMPs it contains. Therefore, we investigated the potential of magnetic manipulation to translocate the BMPNSs to target sites. Initially, a one-dimensional (1-D) model of BMP-NS dynamics was generated under non-flow conditions. The transport of BMP-NSs in a fluid is influenced by both hydrodynamic and magnetic forces. The Newtonian relationship for neurosphere motion with absolute velocity vNS is described as pdNS3 d~ vNS ~ rNS ¼ Fm þ F~d þ F~g ; 6 dt with the magnetic, drag, and gravitational forces on the righthand side.43 In the 1-D model, the gravitational force is not relevant and the inertia force can be neglected with extremely low acceleration. Thus, the magnetic force balances the drag force of the BMP-NS as Fm = Fd mf VBMP Ms;BMP
Fig. 3 (a) Neurosphere culture of control SH-SY5Y cells and BMP-SH cells after incubation for 1 day; D1 and 6 days; D6; (b) confocal image of a BMPNS with cross section view, green: BMPs; red: cell membrane; (c) attachment and spreading of a BMP-NS observed hourly for 8 hours.
morphological features similar to those of spheres formed by the control SH-SY5Y cells without BMP (Fig. 3a). The size of the neurospheres was heterogeneous and varied from 50 to 300 mm
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dHa ¼ 6pgRNS ðvNS vf Þ; dx
where mf is the permeability of the transport fluid, which is similar to the permeability of free space m0: 4p 107, VBMP is the volume of a cluster of BMPs in BMP-NS: 1.68 1018 m3, Ms,BMP is the saturation magnetization of BMPs: 437.90 kA m1, Ha is the externally applied magnetic field intensity at the center of the BMP-NS, g is the fluid viscosity: 0.72 103 kg s1, RNS is the radius of BMP-NS, nNS is the velocity of BMP-NS, and nf is the fluid velocity. The uptake of BMPs in an SH-SY5Y cell was determined to be 8.685 pg per cell (when 20 mg ml1 of BMPs was added to 106 cells; Fig. S1, ESI†), and we assume that the cluster of internalized BMPs is located in the center of a BMP-NS. The magnetic force exerted on a BMP-NS from the media was estimated identically to our previous study.33 The 1-D
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1.77 mm of movement in 60 s, matched well with the experimental data under non-flow conditions.
distribution of Ha is thus described as
Ha
Ms ¼ p
"" 1
tan
#
WL 2xð4x2 þ W 2 þ L2 Þ1=2
Magnetic manipulation of BMP-NSs in an in vitro model system
2 1 6
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tan 4
Ms Ha ¼ p
33 WL
h 2ðx þ TÞ 4ðx þ TÞ2 þ W 2 þ L2
"" 1
tan
#
WL 2xð4x2 þ W 2 þ L2 Þ1=2
2 6 tan1 4
77 i1=2 55
33 WL
77 h i1=2 55; 2 2 2 2ðx þ TÞ 4ðx þ TÞ þ W þ L
where Ms is the saturation magnetization of the NdFeB magnet: 909.46 kA m1, W is the width: 25 mm, and L is length of the magnet: 50 mm. In this non-flow model, RNS was 50 mm and nf is 0. According to the equations, the speed of BMP-NS transport toward the magnet is 29.42 mm s1 when x is 2 mm from the magnet. Fig. 4 shows that a BMP-NS with a radius of 50 mm moved approximately 1.93 mm in 60 s. The theoretical prediction,
Fig. 4 One-dimensional motion of a BMP-NS in non-flow condition under the magnetic field gradient.
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The capture of BMP-NSs at a target site was performed under steady flow conditions. The flow rate was 2 mm s1 in a capillary tube with an internal diameter of 1 mm, which mimics the physiological conditions of rat spinal fluid (Fig. 5a).44 An external magnetic field was applied around the capillary tube using an NdFeB magnet. The intensity of the two-dimensional magnetic field around the capillary tube was evaluated using the COMSOL Multiphysics 3.5 (Fig. 5b). The magnetic flux density decreased rapidly as the distance from the surface of the magnet increased. The magnetic field gradient generated by the NdFeB magnet was approximately B100 T m1 in the capillary region, with the goal of capturing neurospheres with a diameter of B50 mm. The BMP-SH cells and BMP-NSs dispersed in culture medium (104 cells or spheres) were introduced to the capillary using a syringe pump. Effective targeting of the magnetic nanoparticleincorporated cells depended on the magnetic susceptibility of the cells and their linear velocity within the tube. The magnetic susceptibility of the cells was related to the degree of cellular uptake of the magnetic nanoparticles. Interestingly, as shown in Fig. 6a and b, more cells were captured in the target site (magnetic field-focusing region) in the BMP-NSs than in the BMP-SHs, although the amount of BMPs internalized in a BMP-NS and a BMP-SH cell was theoretically identical. A BMP-NS composed of many individual BMP-SH cells, which facilitate to capture more cells at once. Furthermore, BMP-NSs captured earlier on the surface acted as a stopper with a large
Fig. 5 (a) Schematic of in vitro model system to capture BMP-NSs; (b) magnetic flux density on surface (2D surface plot; left) and perimeter of the magnet (graph; right, magnetic flux density at the red line in 2D surface plot).
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Fig. 6 Images of captured BMP-SH cells (a) and BMP-NSs (b) in capillary with 2 mm s1 of flow rate, and images after overnight culture of BMP-SH cells (c) and BMP-NSs (d); red represents PKH26-stained cell membrane.
volume and reduced the speed of the following neurospheres in flow, which were then more easily affected by magnetic force (Fig. S5 and Video, ESI†). These advantages of the neurosphere rendered the capture efficiency of the BMP-NSs to be increased at the target site. The captured cells were incubated overnight in the presence of the magnetic field under normal cell culture conditions, i.e., 37 1C in humidified atmosphere containing 5% CO2. The individual BMP-SH cells and BMP-NSs were well attached to the inner surface of the capillary tube coated with fibronectin after overnight incubation (Fig. 6c). This study confirmed that the BMP-NSs were more effective for transplantation of a large amount of neuronal cells all at once in an in vitro model system.
Conclusions The BMP-NS has the potential to be spatially manipulated by virtue of its free-floating form and the magnetic reactivity of the BMPs it contains, and the BMPs enabled it to attach to the target site. The BMPs also rendered cells to facilitate growth and neuronal differentiation; therefore, BMP-NSs might be suitable for cell therapy for neurological damage. In our approach, focusing of cells at the target site was achieved by BMP loading, and efficiency was improved by delivering the neurosphere form of the cells in vitro. This approach may be useful for stem cell therapy, and future research should apply this technology to in vivo studies.
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Acknowledgements This research was supported by the R&D Program of MKE/KEIT [10033590, Development of Disposable Cancer Diagnosis Sensor of Home-care Type with High Sensitivity], and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A3010079), and by the Brain Korea 21 Plus Project in 2013 (F13SN02D1310), and by MSIP as GFP (CISS-2012M3A6A6054201).
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26 J. Choi, J. Shin, J. Lee and M. Cha, Chem. Commun., 2012, 48, 7474–7476. 27 J. B. Sun, Z. L. Wang, J. H. Duan, J. Ren, X. D. Yang, S. L. Dai and Y. Li, J. Nanosci. Nanotechnol., 2009, 9, 1881–1885. 28 J. Sun, Y. Li, X. J. Liang and P. C. Wang, J. Nanomater., 2011, 2011, 469031–469043. 29 G. Nicolini, M. Miloso, C. Zoia, A. D. Silvestro, G. Cavaletti and G. Tredici, Anticancer Res., 1998, 18, 2477–2482. 30 R. P. Blakemore, D. Maratea and R. S. Wolfe, J. Bacteriol., 1979, 140, 720–729. 31 J.-S. Kim, T.-J. Yoon, K.-N. Yu, M. S. Noh, M. Woo, B.-G. Kim, K.-H. Lee, B.-H. Sohn, S.-B. Park, J.-K. Lee and M.-H. Cho, J. Vet. Sci., 2006, 7, 321–326. 32 T.-G. Iversen, T. Skotland and K. Sandvig, Nano Today, 2011, 6, 176–185. 33 J. Shin, C. H. Yoo, J. Lee and M. Cha, Biomaterials, 2012, 33, 5650–5657. 34 L. Dini and L. Abbro, Micron, 2005, 36, 195–217. 35 K. Sato, H. Yamaguchi, H. Miyamoto and Y. Kinouchi, Biochim. Biophys. Acta, 1992, 1136, 231–238. ¨newa ¨ller, F. Heinzelmann, 36 J. Wiskirchen, E. F. Gro R. Kehlbach, E. Rodegerdts, M. Wittau, H. P. Rodemann, C. D. Claussen and S. H. Duda, Radiology, 2000, 215, 858–862. 37 S. Påhlman, A.-I. Ruusala, L. Abrahamsson, M. E. K. Mattsson and T. Esscher, Cell Differ., 1984, 14, 135–144. 38 J. A. Kim, N. Lee, B. H. Kim, W. J. Rhee, S. Yoon, T. Hyeon and T. H. Park, Biomaterials, 2011, 32, 2871–2877. 39 A. Bez, E. Corsini, D. Curti, M. Biggiogera, A. Colombo, R. F. Nicosia, S. F. Pagano and E. A. Parati, Brain Res., 2003, 993, 18–29. 40 Q. Cao, R. L. Benton and S. R. Whittemore, J. Neurosci. Res., 2002, 68, 501–510. 41 R. Pallini, L. R. Vitiani, A. Bez, P. Casalbore, F. Facchiano, V. Di Giorgi Gerevini, M. L. Falchetti, E. Fernandez, G. Maira, C. Peschle and E. Parati, Neurosurgery, 2005, 57, 1014–1025. 42 S. Pluchino, L. Zanotti, B. Rossi, E. Brambilla, L. Ottoboni, G. Salani, M. Martinello, A. Cattalini, A. Bergami, R. Furlan, G. Comi, G. Constantin and G. Martino, Nature, 2005, 436, 266–271. 43 A. Sinha, R. Ganguly, A. K. De and I. K. Puri, Phys. Fluids, 2007, 19, 117102. 44 R. D. Hazel, E. J. McCormack, J. Miller, J. Li, M. Yu, H. Benveniste, J. P. McAllister II, M. Egnor and M. E. Wagshul, Proceeding of Internal Society for Magnetic Resonance in Medicine, 2006, vol. 14, p. 30.
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PAPER
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Magnetic manipulation of bacterial magnetic nanoparticle-loaded neurospheres† Jaeha Shin,a Kyung-Mee Lee,b Jae Hyup Lee,b Junghoon Lee*ac and Misun Cha*c Specific targeting of cells to sites of tissue damage and delivery of high numbers of transplanted cells to lesion tissue in vivo are critical parameters for the success of cell-based therapies. Here, we report a promising in vitro model system for studying the homing of transplanted cells, which may eventually be applicable for targeted regeneration of damaged neurons in spinal cord injury. In this model system, neurospheres derived from human neuroblastoma SH-SY5Y cells labeled with bacterial magnetic
Received 30th September 2013, Accepted 21st February 2014
nanoparticles were guided by a magnetic field and successfully accumulated near the focus site of
DOI: 10.1039/c3ib40195b
bacterial magnetic nanoparticles to develop successful stem cell targeting strategies during fluid flow,
the magnetic field. Our results demonstrate the effectiveness of using an in vitro model for testing which may ultimately be translated into in vivo targeted delivery of cells through circulation in various
www.rsc.org/ibiology
tissue-repair models.
Insight, innovation, integration We present a promising in vitro model system to study the homing of transplanted stem cells, which may eventually be applicable for targeted regeneration of damaged neurons in spinal cord injury. In this model system, neurospheres were labeled with bacterial magnetic nanoparticles (BMPs) to enhance the homing of transplanted cells to a target site. BMP-loaded neurospheres were guided by a magnetic field and successfully accumulated near the focus site of the magnetic field. This study shows the effectiveness of using an in vitro model for testing BMPs to develop successful stem cell targeting strategies during fluid flow, which may ultimately be translated into in vivo targeted delivery of cells through the circulation in various tissue-repair models.
Introduction The clinical application of stem cells has shown promise for cell transplantation therapies;1–6 however, identifying the optimum delivery strategy of the cells to the injury site remains a major challenge. Targeting and differentiation of stem cells at sites of injury and repair are exciting areas of research in regenerative medicine. Delivery of stem cells to specific targets in vivo has been achieved via direct needle injection to the site of delivery,3,6–9 local infusion,3,10,11 or preferential homing or distribution following systemic administration. However, these
a
School of Mechanical and Aerospace Engineering, Seoul National University, Seoul, 151-744, South Korea b Department of Orthopedic Surgery, College of Medicine, Seoul National University, SMG-SNU Boramae Medical Center, 39 Boramae Gil, Dongjak-Gu, Seoul, 156-707, South Korea c Inter-University Semiconductor Research Center, Seoul National University, Seoul, 151-744, South Korea. E-mail: [email protected], [email protected]; Fax: +82-2-877-9104; Tel: +82-2-880-9101 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3ib40195b
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delivery methods generally suffer from a lack of precise localization, low repeatability, and local damage to the injection site. The versatile intrinsic properties of magnetic particles render them useful for targeted cell therapy, where magnetic carriers loaded with cells can be directed or guided by means of a magnetic field gradient toward biological targets.12,13 Recent studies have shown that magnetic labeling of cells is relatively easy and safe.14–18 However, only a few studies have used magnetic fields to guide magnetic particle-loaded cells to specific targets because of low uptake at the site of injury.19,20 Herein, we present a promising model system that uses magnetic particle loading of cell spheres to enhance the homing of transplanted cells to a target site; this system may therefore be useful for facilitating the regeneration of damaged neurons in spinal cord injury. The bacterial magnetic nanoparticles (BMPs) employed in our study are inherently biocompatible and disperse well in aqueous solutions because of a stable lipid membrane wrapping the magnetic core.21–23 This lipid membrane contains lipids and proteins, such as phosphatidylserine, phosphatidylethanolamine, glycolipids, and sulfolipids.21–23 In spite of concerns about embedded proteins of lipid membrane, the BMPs showed no significant toxicity in vitro and in vivo.24–28
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BMP-loaded neurospheres (BMP-NSs) of SH-SY5Y cells, a cell line commonly used for in vitro neurological studies,29 were investigated with regard to targeted delivery and transplantation into the lesion site in an in vitro model of the spinal cord. Cells delivered in the spheres demonstrated improved delivery efficiency relative to individual cells delivered in suspension.
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Materials and methods BMP preparation BMPs were extracted from the magnetotactic bacterium (MTB) Magnetospirillum sp. AMB-1 (ATCCs 700264), which was cultured in magnetic spirillum growth medium (MSGM) for 5 d in a shaking incubator at 30 1C under anaerobic conditions.30 After sufficient culture, the cells were centrifuged at 5000 rpm for 25 min and were then lysed using a homogenizer (VCX500, Sonics & Materials, USA) for 30 min. BMPs were collected using a neodymium iron boron (NdFeB) magnet and washed five times with 1 PBS. The BMPs collected were dispersed in 1 PBS and sterilized by autoclaving (121 1C, 15 min). The concentration of the extracted BMPs was determined by inductive coupled plasma-atomic emission spectrometry (ICP-AES; ICPS-7500, Shimadzu, Japan).
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cacodylate buffer for 2 h at 4 1C. The cells were then briefly washed twice with distilled water, stained with 0.5% uranyl acetate for 30 min at 4 1C, and dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, and 100%) for 10 min each. The samples were treated with 100% propylene oxide twice for 10 min and embedded in Spurr’s resin. Ultrathin sections were prepared using an ultramicrotome (MT-X, RMC, USA) and mounted on copper grids. The sections were examined using a TEM (JEM-1010, JEOL, Japan). BMPs were labeled with fluorescein isothiocyanate (FITC) to allow tracking of the location of internalized BMPs in the cells using confocal laser scanning microscopy. After delivery of FITC-labeled BMPs, the cells were fixed with 4% paraformaldehyde in 1 PBS for 15 min at room temperature. After two washes with wash buffer, i.e., 0.1% Triton X-100 in 1 PBS, the fixed cells were permeabilized and blocked using 1% BSA and 0.1 Triton X-100 in 1 PBS for 20 min. Actin filaments were then stained with TRITC-conjugated phalloidin (1 : 200) for 50 min at room temperature. The cells were briefly washed three times with 1 PBS, and the DNA in the nuclei was counterstained using dilute DAPI in 1 PBS (1 : 1000) applied three times for 5 min each. The stained cells were examined using a confocal laser scanning microscope (LSM710, Carl Zeiss, Germany).
Cell culture and intracellular uptake of BMPs SH-SY5Y cells, a human-derived neuroblastoma cell line, were cultured in Dulbecco’s modified Eagle medium (DMEM; Thermo Scientific, USA) containing 10% (v/v) fetal bovine serum (FBS; Thermo Scientific, USA) and 1% penicillin/streptomycin (Thermo Scientific, USA) at 37 1C in a humidified atmosphere containing 5% CO2. The cells were cultured to 80% confluency in BMP-free culture medium and were then treated with BMPs (10 mg ml1 applied to more than 106 cells) in serum-reduced medium (1% FBS) and incubated overnight to allow internalization of BMPs. The BMP-loaded cells (BMP-SH) were separated from non-BMPloaded cells using a magnet. For neurosphere formation, the cells were cultured in a floating culture for 5 d. Cell staining The cell membrane was labeled with red fluorescent dye (PKH26, Sigma-Aldrich, USA) for tracking of single cells and neurospheres in the target site; individual cells were cultured under appropriate conditions for subsequent experiments and neurospheres were formed. Stained cells were examined using a fluorescence microscope (Nikon, Japan). Microscopy Transmission electron microscope (TEM) imaging of the cells was performed after overnight treatment with the BMPs to confirm internalization of the BMPs. The treated cells were immersed in modified Karnovsky’s fixative solution (2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate buffer) for 4 h at 4 1C. After three washes for 10 min in 0.05 M sodium cacodylate buffer, the cells were post-fixed with 1% osmium tetroxide in 0.05 M sodium
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Exposure of static magnetic field Static magnetic fields (SMFs) were generated with a neodymium iron boron (NdFeB) magnet (50 25 25 mm) with an average flux density of 480 10 mT, as measured by a Gauss meter (TM-701, Kanetec, Japan). Magnets were placed under the plastic culture dishes when changes in cell growth were evaluated. Because the SMF intensity decreases according to the square of the distance from the magnet, the magnetic force on the cells was calculated taking the thickness of the bottom of the culture dish (1.0 mm) into account. The SMF was applied continuously at the bottom of the culture dish for up to 6 d. When neurite outgrowth was measured, the magnets were placed laterally on both sides of the culture dish (Fig. S2, ESI†), and the cells were treated with retinoic acid (RA, all trans-retinoic acid; Sigma, USA) for 1 week. MTS assay The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed to evaluate the cytotoxicity of the BMPs and viability of the cells. The cells (5 103 cells per well) were seeded on a 96-well plate with 120 ml of culture medium. After the cells were sufficiently cultured, 25 ml of CellTiter 96s AQueous One Solution Reagent (Promega, USA) was added into each well. The plate was then incubated at 37 1C for 1–4 h in a humidified 5% CO2 atmosphere, and the absorbance was recorded at 490 nm using a microplate reader (Sunrise, Tecan, Switzerland). All the assays were performed in three separate sets of experiments, and the absorbance of control cells was defined as 100% viability in every assay.
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Measurement of neurite outgrowth Differentiation of SH-SY5Y and BMP-SH cells was induced by treating them with 10 mM retinoic acid (RA) in serum-reduced medium; the medium was changed every 3 d. Neurite outgrowth of the cells was assessed after 7 d of differentiation with or without exposure to SMF. Neurite length was measured and classified into three levels according to the length, i.e., 0–50 mm, 50–100 mm, or over 100 mm. In total, 200 cells in each condition were analyzed in three separate sets of the experiments, with the standard error of the mean being recorded. Manipulation of BMP-loaded cells and neurospheres BMP-loaded individual SH-SY5Y cells (BMP-SHs) and BMP-NSs were prepared as described above and labeled with a red fluorescent dye to track the movement. In the non-flow condition, the magnetic field was produced using an NdFeB magnet and the motion of the BMP-NSs was observed within 2 mm from the magnet, where the field gradient was approximately 40 T m1. To study the delivery of BMP-SHs and BMP-NSs into a simulated spinal cord under flow conditions, a glass capillary tube with a diameter of 1 mm was employed. The inner surface of the tube was coated with fibronectin to permit attachment of the cells on the surface after delivery. The sterilized capillary tube was filled with a solution of 100 mg ml1 fibronectin in 1 PBS, incubated overnight at room temperature, and then dried. A syringe pump (KD Scientific, USA) was used to introduce the cells dispersed in culture medium (104 cells or spheres) into the capillary tube with a stable flow rate of 22.6 ml h1. As the cells flowed in the tube, a focused magnetic field was applied to a target point to capture the cells using an edge of the magnet that generated approximately 100 T m1 of field gradient. The capillary tube filled with the culture medium and the captured cells were incubated at 37 1C for 20 h in a humidified 5% CO2 atmosphere after delivery of the cells. The movement of the BMP-SHs and BMP-NSs under flow conditions and attachment of the cells on the inner surface of the tube were examined using a fluorescence microscope (Nikon, Japan).
Results and discussion
Fig. 1 Observation of BMPs internalized SH-SY5Y cells using (a) confocal microscopy; (b) TEM of the cell, scale bars: 10 mm, red: actin filaments, blue: nucleus, green: BMPs; (c) cytotoxicity of BMPs to SH-SY5Y cells with various concentrations with standard error of the mean (SEM) and p-value from unpaired t-test.
Uptake and cytotoxicity of BMPs Magnetospirillum sp. AMB-1 is an MTB that is widely used because it grows well under laboratory conditions. BMPs from AMB-1 contain a well-crystallized magnetite (Fe3O4) core and an enveloping magnetosome membrane that has a lipid bilayer and embedded proteins.21–23 These BMPs are highly crystallized, with few defects, a uniform size, and ferrimagnetic properties that are difficult to achieve in synthesized magnetic nanoparticles at room temperature.21–23 The uptake of BMPs by SH-SY5Y cells was directly confirmed by electron microscopy. The BMPs were labeled with FITC to track the location of internalized BMPs by using confocal laser scanning microscopy. As shown in Fig. 1a, the BMPs were internalized well into the SH-SY5Y cells, and most of the internalized BMPs were found in the cytosol. The TEM images
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clearly showed that the internalized BMPs were mostly located in the endosome or lysosome in the cytosol; a few BMPs escaped from the endosome were found in the cytosol after overnight uptake (Fig. 1b, right upper and lower boxes). Endocytosis is a well-known mechanism for cellular uptake of nanoparticles, and most of the internalized particles are typically localized in the endosome or lysosome.31,32 The uptake efficiency of the BMPs in SH-SY5Y cells was 43.42% when BMPs at a concentration of 20 mg ml1 were delivered to 106 cells (Fig. S1, ESI†); this value is comparable with the uptake of BMPs by HeLa cells,33 as evaluated by measuring the ferric concentration using ICP-AES (ICPS-7500, Shimadzu, Japan). BMPs showed no cytotoxicity against SH-SY5Y cells up to a concentration of 300 mg ml1 for 5000 cells
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during culture for 3 d. The viability of the cells decreased at BMP concentrations over 75 mg ml1 after 6 d of incubation; therefore, SH-SY5Y cells were treated with BMP concentrations lower than 75 mg ml1 in subsequent studies (Fig. 1c).
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Enhancement of cell growth and neuronal differentiation with internalization of BMPs In a previous study,33 we reported the in vitro biological effects of the communications between internalized BMPs and an external SMF. Combination of the BMPs and SMF induced modulation of GPCRs-mediated signal transduction, thereby alteration of cell structure and the enhanced cell growth were caused. Also, an antiapoptotic effect was induced by the synergetic interaction of magnetism. In this study, the magnetic response of BMP-SH cells was investigated under continuous exposure to an SMF. The SMF (480 10 mT) was constantly applied to investigate cell growth at the bottom of the dish for up to 6 d. However, the synergetic effect of the BMPs and SMF was not observed in SH-SY5Y cells. Internalization of BMPs induced a substantial increase in SHSY5Y cell growth (up to 30% in 6 days), whereas the effect of SMF exposure was negligible regardless of BMP internalization (Fig. 2a). MFs are thought to influence the viability of cells because of the diamagnetic properties of the molecules in the cell membrane.34 Some types of cells, however, have been reported to be essentially unaffected by exposure to MFs,35,36 such as the SH-SY5Y cells. Neuronal differentiation of the BMP-SH cells was induced by constant treatment with RA (10 mM ml1) for 7 d under SMF conditions. The SMF (100–220 mT) was applied to cells laterally as neuronal differentiation was induced by RA treatment (Fig. S2 and S3, ESI†). The neurite length was measured and classified according to three levels of length, i.e., 0–50 mm, 50–100 mm, or over 100 mm, after 7 d of differentiation (Fig. 2b and c). The length of the neurites was significantly higher in BMP-SH cells (regardless of their exposure to SMF) than in BMP-free cells, particularly with regard to the ratio of neurites longer than 50 mm (Fig. 2b and c). Neurite outgrowth is a major morphological alteration in neuronal differentiation induced by RA.37 Improvement of neurite outgrowth using magnetic nanoparticles has been previously reported in another neural cell line, PC12 cells.38 It has been proposed that magnetic nanoparticles could be exploited to enhance or accelerate nerve regeneration and to provide guidance for regenerating axons.38 Nanoparticles may also be used to create mechanical tension to stimulate the growth and elongation of axons. Our results imply that targeted magnetic cell delivery to injury sites without side effects may be possible using SMF and that BMP-SH cells delivered to lesion sites magnetically may afford efficient regeneration and healing. BMP-loaded neurosphere culture A neurosphere is a free-floating spherical cluster of neural stem cells obtained from floating cultures in vitro.39 Neurospherederived cells have been successfully used in models of
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Fig. 2 (a) Cell growth of SH-SY5Y cells under an SMF with SEM and pvalue from unpaired t-test (**p o 0.005, ***p o 0.0001), (b) measurement of neurite outgrowth length and (c) microscope images of SH-SY5Y cells with treatment of RA (10 mM) under an SMF, scale bars: 50 mm.
neurological injury such as experimental autoimmune encephalomyelitis and spinal cord injury.40–42 Therefore, neurospheres may be useful for the delivery of therapeutic cells to lesion sites. SH-SY5Y human neuroblastoma cells can grow in neurospheres under floating culture conditions without requiring special treatment such as serum and growth factors. We confirmed that the BMP-SH cells formed neurospheres with
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after 5–10 d of free-floating culture. Also, as shown in Fig. 3b, the green spots indicating FITC-labeled BMPs were maintained in the cells as they grew into neurospheres. The neurospheres formed from single BMP-SH cells theoretically have the same amount of BMPs found in individual BMP-SH cells. Moderately grown neurospheres (diameter, B100 to 200 mm) were isolated and cultured on an extracellular matrix (ECM)coated surface to investigate transplantation of neurospheres after delivery to the target site. The neurospheres attached to the surface, and individual cells from the neurospheres migrated to the surrounding area (Fig. 3c) in similar patterns as control cells (Fig. S4, ESI†). The results show that the BMP-NSs could be applied for delivery of therapeutic cells with similar functionality of neurospheres from control cells. Theory of manipulation of BMP-NSs The BMP-NS has the potential to be spatially manipulated by virtue of its free-floating form and the magnetic reactivity of the BMPs it contains. Therefore, we investigated the potential of magnetic manipulation to translocate the BMPNSs to target sites. Initially, a one-dimensional (1-D) model of BMP-NS dynamics was generated under non-flow conditions. The transport of BMP-NSs in a fluid is influenced by both hydrodynamic and magnetic forces. The Newtonian relationship for neurosphere motion with absolute velocity vNS is described as pdNS3 d~ vNS ~ rNS ¼ Fm þ F~d þ F~g ; 6 dt with the magnetic, drag, and gravitational forces on the righthand side.43 In the 1-D model, the gravitational force is not relevant and the inertia force can be neglected with extremely low acceleration. Thus, the magnetic force balances the drag force of the BMP-NS as Fm = Fd mf VBMP Ms;BMP
Fig. 3 (a) Neurosphere culture of control SH-SY5Y cells and BMP-SH cells after incubation for 1 day; D1 and 6 days; D6; (b) confocal image of a BMPNS with cross section view, green: BMPs; red: cell membrane; (c) attachment and spreading of a BMP-NS observed hourly for 8 hours.
morphological features similar to those of spheres formed by the control SH-SY5Y cells without BMP (Fig. 3a). The size of the neurospheres was heterogeneous and varied from 50 to 300 mm
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dHa ¼ 6pgRNS ðvNS vf Þ; dx
where mf is the permeability of the transport fluid, which is similar to the permeability of free space m0: 4p 107, VBMP is the volume of a cluster of BMPs in BMP-NS: 1.68 1018 m3, Ms,BMP is the saturation magnetization of BMPs: 437.90 kA m1, Ha is the externally applied magnetic field intensity at the center of the BMP-NS, g is the fluid viscosity: 0.72 103 kg s1, RNS is the radius of BMP-NS, nNS is the velocity of BMP-NS, and nf is the fluid velocity. The uptake of BMPs in an SH-SY5Y cell was determined to be 8.685 pg per cell (when 20 mg ml1 of BMPs was added to 106 cells; Fig. S1, ESI†), and we assume that the cluster of internalized BMPs is located in the center of a BMP-NS. The magnetic force exerted on a BMP-NS from the media was estimated identically to our previous study.33 The 1-D
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1.77 mm of movement in 60 s, matched well with the experimental data under non-flow conditions.
distribution of Ha is thus described as
Ha
Ms ¼ p
"" 1
tan
#
WL 2xð4x2 þ W 2 þ L2 Þ1=2
Magnetic manipulation of BMP-NSs in an in vitro model system
2 1 6
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tan 4
Ms Ha ¼ p
33 WL
h 2ðx þ TÞ 4ðx þ TÞ2 þ W 2 þ L2
"" 1
tan
#
WL 2xð4x2 þ W 2 þ L2 Þ1=2
2 6 tan1 4
77 i1=2 55
33 WL
77 h i1=2 55; 2 2 2 2ðx þ TÞ 4ðx þ TÞ þ W þ L
where Ms is the saturation magnetization of the NdFeB magnet: 909.46 kA m1, W is the width: 25 mm, and L is length of the magnet: 50 mm. In this non-flow model, RNS was 50 mm and nf is 0. According to the equations, the speed of BMP-NS transport toward the magnet is 29.42 mm s1 when x is 2 mm from the magnet. Fig. 4 shows that a BMP-NS with a radius of 50 mm moved approximately 1.93 mm in 60 s. The theoretical prediction,
Fig. 4 One-dimensional motion of a BMP-NS in non-flow condition under the magnetic field gradient.
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The capture of BMP-NSs at a target site was performed under steady flow conditions. The flow rate was 2 mm s1 in a capillary tube with an internal diameter of 1 mm, which mimics the physiological conditions of rat spinal fluid (Fig. 5a).44 An external magnetic field was applied around the capillary tube using an NdFeB magnet. The intensity of the two-dimensional magnetic field around the capillary tube was evaluated using the COMSOL Multiphysics 3.5 (Fig. 5b). The magnetic flux density decreased rapidly as the distance from the surface of the magnet increased. The magnetic field gradient generated by the NdFeB magnet was approximately B100 T m1 in the capillary region, with the goal of capturing neurospheres with a diameter of B50 mm. The BMP-SH cells and BMP-NSs dispersed in culture medium (104 cells or spheres) were introduced to the capillary using a syringe pump. Effective targeting of the magnetic nanoparticleincorporated cells depended on the magnetic susceptibility of the cells and their linear velocity within the tube. The magnetic susceptibility of the cells was related to the degree of cellular uptake of the magnetic nanoparticles. Interestingly, as shown in Fig. 6a and b, more cells were captured in the target site (magnetic field-focusing region) in the BMP-NSs than in the BMP-SHs, although the amount of BMPs internalized in a BMP-NS and a BMP-SH cell was theoretically identical. A BMP-NS composed of many individual BMP-SH cells, which facilitate to capture more cells at once. Furthermore, BMP-NSs captured earlier on the surface acted as a stopper with a large
Fig. 5 (a) Schematic of in vitro model system to capture BMP-NSs; (b) magnetic flux density on surface (2D surface plot; left) and perimeter of the magnet (graph; right, magnetic flux density at the red line in 2D surface plot).
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Fig. 6 Images of captured BMP-SH cells (a) and BMP-NSs (b) in capillary with 2 mm s1 of flow rate, and images after overnight culture of BMP-SH cells (c) and BMP-NSs (d); red represents PKH26-stained cell membrane.
volume and reduced the speed of the following neurospheres in flow, which were then more easily affected by magnetic force (Fig. S5 and Video, ESI†). These advantages of the neurosphere rendered the capture efficiency of the BMP-NSs to be increased at the target site. The captured cells were incubated overnight in the presence of the magnetic field under normal cell culture conditions, i.e., 37 1C in humidified atmosphere containing 5% CO2. The individual BMP-SH cells and BMP-NSs were well attached to the inner surface of the capillary tube coated with fibronectin after overnight incubation (Fig. 6c). This study confirmed that the BMP-NSs were more effective for transplantation of a large amount of neuronal cells all at once in an in vitro model system.
Conclusions The BMP-NS has the potential to be spatially manipulated by virtue of its free-floating form and the magnetic reactivity of the BMPs it contains, and the BMPs enabled it to attach to the target site. The BMPs also rendered cells to facilitate growth and neuronal differentiation; therefore, BMP-NSs might be suitable for cell therapy for neurological damage. In our approach, focusing of cells at the target site was achieved by BMP loading, and efficiency was improved by delivering the neurosphere form of the cells in vitro. This approach may be useful for stem cell therapy, and future research should apply this technology to in vivo studies.
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Acknowledgements This research was supported by the R&D Program of MKE/KEIT [10033590, Development of Disposable Cancer Diagnosis Sensor of Home-care Type with High Sensitivity], and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A3010079), and by the Brain Korea 21 Plus Project in 2013 (F13SN02D1310), and by MSIP as GFP (CISS-2012M3A6A6054201).
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