ARTICLE pubs.acs.org/Langmuir
A Direct Technique for Magnetic Functionalization of Living Human Cells Maria R. Dzamukova,†,§ Alsu I. Zamaleeva,†,§,|| Dilara G. Ishmuchametova,† Yuri N. Osin,† Andrey P. Kiyasov,‡ Danis K. Nurgaliev,† Olga N. Ilinskaya,† and Rawil F. Fakhrullin*,† †
Biomaterials and Nanomaterials Group, Faculty of Biology and Soil, Kazan (Idel buye/Volga region) Federal University, Kreml uramı 18, Kazan, Republic of Tatarstan, 420008, RF ‡ Department of Normal Anatomy, Kazan State Medical University, Butlerov uramı 49, Republic of Tatarstan, 420012, RF
bS Supporting Information ABSTRACT: Functionalized living cells are regarded as effective tools in directed cell delivery and tissue engineering. Here we report the facile functionalization of viable isolated HeLa cells with superparamagnetic cationic nanoparticles via a single-step biocompatible process. Nanoparticles are localized on the cellular membranes and do not penetrate into the cytoplasm. The magnetically responsive cells are viable and able to colonize and grow on substrates. Magnetically facilitated microorganization of functionalized cells into viable living clusters is demonstrated. We believe that the technique described here may find a number of potential applications in cell-based therapies and in development of whole-cell biosensors.
’ INTRODUCTION Cell therapy aims to treat the disease by replacing damaged or diseased cells in tissues and organs1 with isolated normal cells (including stem cells), focusing the attention of researchers on novel ways of effective and controllable delivery and spatial organization of cells. Functionalization of cells with nanoscale magnets makes them magnetically responsive, i.e. the functionalized cells can be spatially manipulated with an external magnetic field. 2 Magnetic nanoparticles surface-modified with receptor-specific ligands3 6 can be applied for capturing and isolation of cancer cells from bodily fluids in vivo, which reduces metastasis and impedes tumor progress.7 Studying of interactions of living cells with superparamagnetic iron oxide nanoparticles (SPIONs) has recently gained considerable attention.2,8 10 The main advantage of magnetically functionalized cells is that they can be distantly accumulated and collected from multicomponent mixtures,11 positioned inside microchannels12 and assembled on electrodes,13 thus providing a versatile means of control over cells distribution and immobilization. Potentially, magnetically functionalized cells introduced into a more complex system (i.e., living tissue or organ) can be similarly manipulated with an external magnetic field and deliberately positioned and concentrated within a certain area. If applied to humans, this would considerably contribute to the development of cell therapy methods. However, this requires the biocompatible functionalization of human cells, preserving the membrane integrity and ability for cell division. It has been already demonstrated that cell-wall-enclosed microorganisms can be functionalized r 2011 American Chemical Society
with SPIONs.2,9,11 However, human cells lack any protective envelope except the fragile cellular membrane, which can be easily destroyed even by the osmotic pressure when the cells are transferred from the isotonic media into water. This makes the direct deposition of SPIONs with simultaneous preserving of cellular viability very difficult, because nanoparticles tend to aggregate in buffer solutions. Therefore, the known techniques of human cells labeling with magnetic nanoparticles6 are based on grafting of nanoparticles with antibodies,5 aptamers,3 polypeptides and proteins,4 or magnetoliposomes,14,15 which results in accumulation of nanoparticles inside the cells. These techniques are effective for labeling and isolation of cells in vitro; however, they are elaborate, expensive, and time-consuming and require specific affinity moieties to be attached to the SPIONs. This makes them inappropriate in magnetic functionalization of isolated individual human cells, if the magnetically responsive viable cells need to be delivered into a certain place and immobilized using an external magnetic field. Therefore, it is challenging to establish a novel strategy of magnetic functionalization, when the isolated human cells are rapidly surfacefunctionalized with SPIONs via a single-step facile process. Here we demonstrate a robust and direct technique for the magnetic functionalization of human cells based on polyelectrolyte-stabilized SPIONs. Cell surfaces of isolated HeLa cells were coated Received: September 30, 2011 Revised: October 25, 2011 Published: October 27, 2011 14386
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Langmuir with SPIONs, which can be further organized in multicellular clusters using an external magnetic field. Importantly, the functionalization strategy shown here is biocompatible, i.e., functionalized HeLa cells remain viable and able to colonize and grow on appropriate substrates.
’ MATERIALS AND METHODS Synthesis and Characterization of SPIONs. SPIONs were prepared as reported elsewhere,8 with some alterations: 2.0 mL of 1 M FeCl3 (Sigma) and 0.5 mL of 2 M FeCl2 (Sigma) aqueous solutions were mixed and stirred vigorously, and then 25 mL of 0.5 M aqueous NaOH was added dropwise while the mixture stirred, resulting in the formation of iron oxide precipitate. The precipitate was then separated using a magnet and washed extensively with water until the supernatant reached pH 7. To stabilize the as-made SPIONs with the cationic polyelectrolyte, 1 mL of the nanoparticles was added into 10 mL of 10 mg mL 1 aqueous PAH, the mixture was sonicated for 10 min (Bandelin Sonoplus sonifier) and then separated by centrifugation, and the solids were washed with Milli-Q water five times. SPIONs were filtered using a 220 nm pore size syringe filter, which eliminated larger aggregates and sterilized the suspension. The size distribution of the resulting MNPs was determined from TEM and SEM (see the detailed description of the instruments below). Hydrodynamic diameters and ζ-potentials were measured using a Malvern Zetasizer Nano ZS instrument and standard cells. The magnetic properties of the samples of PAH-stabilized SPIONs were investigated using a homemade coercivity spectrometer. The samples were placed onto the nonmagnetic paper sample holders and the isothermal hysteresis loops were acquired at 300 K. Flow-injection study of resonant frequency (ΔF) and motional resistance (ΔR) changes during adsorption of SPIONs were studied using a QCM 200 (SRS) device and the flow-injection setup as described previously.16 Briefly, 5 MHz AT-cut quartz crystals (SRS) with gold electrodes were mounted in an axial flow-cell, and the crystal was exposed by only one electrode to the flowing solution. A peristaltic pump was used to produce 80 μL min 1 flow rate at room temperature. ΔF and ΔR shifts were recorded simultaneously as a function of time using LabVIEW StandAlone Software (National Instruments). HeLa Cell Culture. HeLa cells were cultured in plastic cell culture plates (growth area 9.4 cm2) at 37 °C in humidified atmosphere of 5% CO2 in Dulbecco’s modified Eagle media (DMEM) (Invitrogen) containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin (100 U mL 1/100 μg mL 1). Cells were grown to 70 80% confluency and then they were washed with Dulbecco’s phosphate buffered saline (DPBS) and detached from the wells using 0.25% trypsin EDTA (Sigma) during 5 min incubation followed by centrifugation (500 g) and washing with DPBS. Functionalization of HeLa Cells with SPIONs. HeLa cells (5 106 mL 1) in DPBS were dropwise introduced into sterile SPIONs suspension (0.6 mg mL 1) in 0.15 M NaCl. After 3 min incubation with gentle vortexing, the cells were separated from the remaining SPIONs using a permanent neodymium iron boron (NdFeB) magnet and washed three times with DPBS. To demonstrate the magnetically facilitated microorganization of SPION-functionalized cells into multicellular clusters, we deposited 2 mm NdFeB cylindrical magnets below the culture wells, which were then inoculated with SPION-functionalized HeLa cells (3 105 mL 1) Viability Tests. The membrane integrity of intact and SPIONfunctionalized HeLa cells was tested using LIVE/DEAD viability/ cytotoxicity kit L-70-13 (Molecular probes) according to the manufacturer’s protocol.17 The enzymatic activity of intact and SPION-functionalized HeLa cells was tested using fluoresceine diacetate (FDA) as described elsewhere.18 Fluorescence microscopy was used to estimate the viability in the stained samples. Flow cytometry was employed along with FDA staining to determine the percentage of viable cells in the
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samples. A FACSCalibur (Becton Dickinson) instrument was used. Data was obtained using Cell Quest Pro software. Charaterization Techniques. Optical and fluorescence microscopy images were obtained using a Carl Zeiss AxioScope A1 microscope equipped with an AxioCam MRc5 CCD camera. Cell culture wells were examined using a Leica DMIL inverted microscope equipped with a Leica DFC 420 CCD camera. Real-time footage was obtained using a Carl Zeiss Jenamed microscope equipped with a Webbers M320 CCD camera. TEM images of the thin-sectioned SPION-functionalized HeLa cells were obtained using a JEOL 1200 EX microscope operating at 80 kV. The cells were fixed with 2.5% glutaraldehyde, gradually dehydrated using a series of ethanol solutions (30, 60, 70, 80, and 100%), embedded into Epon resin, and then thin sections were cut using a LKB ultramicrotome equipped with a diamond knife and mounted on copper grids. The samples were stained with 2% aqueous uranyl acetate and lead citrate. SEM images and EDX spectra and mapping images were obtained using an Auriga (Carl Zeiss) instrument equipped with an Inca Energy 350 X-Max (Oxford Instruments) spectrometer. Cells were dehydrated as described above and placed on glass stubs. Samples were sputter-coated with Au (60%) and Pd (40%) alloy using a Q150R (Quorum Technologies) instrument. Images were obtained at 3 10 4 Pa working pressure and 15 keV accelerating voltage using InLens detection mode (2 mm working distance). Spectra acquisition time in EDX mapping mode was 30 min.
’ RESULTS AND DISCUSSION We synthesized and used throughout (poly)allylamine hydrochloride (PAH) stabilized positively charged SPIONs. The synthesis procedure is based on coprecipitation of Fe2+ and Fe3+ ions in alkali media and yields in a mixture of almost spherical iron oxide nanoparticles. We used a polycation (PAH) to stabilize the iron oxide nanoparticles, which are poorly stable in water, and to provide them with a positive surface charge. SPIONs were introduced into a concentrated aqueous solution of PAH, sonified, collected with a permanent magnet, and then washed several times with water to remove the remaining free PAH from the suspension. The process does not require additional purification steps using toxic solvents, as in oleatemediated SPIONs synthesis.19 Transmission (TEM) and scanning (SEM) electron microscopy were used to characterize the SPIONs obtained; as one can see in Figure 1A,B, a fairly monodisperse suspension of magnetic nanoparticles with an average diameter of around 15 nm was produced. Hydrodynamic diameters of the SPIONs in water were around 100 nm (Figure 1C). The colloid stability of aqueous SPIONs after filtration was preserved for at least 3 months at room temperature. Stabilization with PAH rendered the SPIONs with a positive ζ-potential (34 ( 7 mV). Isothermal superparamagnetic magnetization properties of the SPIONs are shown in Figure 1D. Quartz crystal microbalance (QCM) microgravimetry was applied to study the adsorption behavior of PAH-stabilized SPIONs. Gold electrode surface of a quartz crystal was used to mimic the deposition of SPIONs onto the cell membranes via simultaneous monitoring of frequency and motional resistance shifts in a flow-injection mode. As seen in Figure 1E, SPIONs (0.6 mg mL 1) adsorbed irreversibly during 7 min after injection into the flow chamber and were not removed during washing with water. This demonstrates that the PAH-stabilized SPIONs strongly adhere to negatively charged surfaces (i.e., cell membrane) and the adsorption occurs rapidly, which indicates that the exposure time could be maximally reduced if applied to cells. 14387
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Figure 1. Structural and magnetic properties of PAH-stabilized SPIONs. (A) TEM and (B) SEM image of SPIONs. (C) distribution of hydrodynamic diameters of PAH-stabilized SPIONs in water. (D) Room-temperature magnetization versus applied magnetic field curve for the SPIONs demonstrating their superparamagnetic behavior. (E) QCM resonant frequency (ΔF) and motional resistance (ΔR) changes during adsorption of SPIONs onto gold electrodes.
Figure 2. Bright-field optical microscopy images of (A, B) intact HeLa cells; (C) fluorescence microscopy image of FITC PAH-coated HeLa cells; (D) a merged bright-field and fluorescence microscopy image of a single HeLa cell modified with FITC PAH; (E, F) bright-field optical microscopy images of SPION-functionalized HeLa cells (note the characteristic brown color of the cells due to SPIONs’ deposition).
We have chosen HeLa cells (Figure 2A,B) as a model for magnetic functionalization with cationic SPIONs. Cells were detached from the culture dishes using trypsin EDTA and suspended in DPBS. HeLa cells possess a low negative surface charge (ζ-potential of 20 ( 3 mV); therefore, cationic polymers (i.e., FITC PAH) easily attach to their membranes (Figure 2C,D). One can see the uniform FITC PAH fluorescence localized on the cellular membranes, suggesting that the cationic SPIONs can be deposited in a similar way. The one-step magnetic functionalization process which we employed here is very simple and fast. Harvested cells were transferred from the
plates into suspension and then introduced into the saline suspension of SPIONs (0.6 mg mL 1) and incubated for 5 min while gently shaking. Then the cells were separated from the remaining solution with a permanent magnet and washed with DPBS. The optical microscopic examination of the cells demonstrated that they are coated with a uniform layer of SPIONs. The color of the cells has been changed drastically (Figure 2E,F) in comparison with the native cells. The similar visible change of color in functionalized cells was reported previously with yeast.2 The SPION-functionalized HeLa cells were magnetically responsive and could be collected or spatially moved with a 14388
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Figure 3. SEM images (at increasing magnifications) of (A, B) intact Hela cells and (C, D) SPION-functionalized Hela cells. EDX characterization of SPION-functionalized HeLa cells: (E) SEM image and (F) corresponding iron distribution map of a single SPION-functionalized cell. (G) EDX spectrum obtained from a single SPION-functionalized cell (inset), showing characteristic Fe peaks.
Figure 4. TEM images (at increasing magnifications) of (A and C) intact Hela cells and (B and D) SPION-functionalized Hela cells. 14389
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Langmuir permanent magnet, as shown in movie 1 of the Supporting Information. As expected, SPION-functionalized HeLa cells exhibited superparamagnetic behavior (Supporting Information, Figure S1). SEM images (Figure 3) also confirmed the uniform deposition of SPIONs on the cells, indicating the random distribution of nanoparticles on and between the elaborate microvilli network. High-magnification SEM images demonstrate that the nanoparticles are concentrated as a monolayer on the surface of the cell. Energy-dispersive X-ray (EDX) spectroscopy was further employed to demonstrate the deposition of SPIONs, and the results (EDX spectra and Fe mapping) are given in Figure 3E G). The corresponding EDX spectra of an intact HeLa cell is presented in Figure S2, Supporting Information, where no iron peaks are distinguishable. Interestingly, we found that the blebs (spherical cytoplasm protrusions of various diameters) were not coated with nanoparticles (Figure S3, Supporting Information). This further indicates, that SPIONs are concentrated around microvilli, do not penetrate into cytoplasm, and can be shifted from a certain region during the formation of blebs. Transmission electron microscopy (TEM) was further employed to study in more detail the deposition of SPIONs on the HeLa cells. The typical TEM images of intact and SPIONfunctionalized cells are given in Figure 4. The worm-like protrusions seen around the cell body are the ultracut microvilli projections characteristic to TEM images of isolated HeLa cells. TEM observations confirm that the SPIONs are predominantly located around micrometer-long microvilli (Figure 3D). In TEM images we did not notice the penetration of the SPIONs into the cytoplasm, which can be explained by the preferential attachment of the positively charged SPIONs to anionic carbohydrates constituting the outer layer of micrivilli. On the contrary, anionic magnetic nanoparticles are known to internalize within cellular endosomes.20 Overall, the microscopic study of the SPION-functionalized cells allowed us to conclude that (i) HeLa cells are efficiently coated with a uniform layer of magnetic nanoparticles which (ii) do not pierce the cellular membrane. The absence of SPIONs in cytoplasm indicates that the magnetic functionalization with PAH-stabilized nanoparticles is a highly biocompatible process. Indeed, previously we have shown that PAH-coated SPIONs are not toxic if used to functionalize yeast,2,7 microalgae,8 bacteria,11 and nematodes.21 Human cells, however, are much more vulnerable in comparison with microbes, since they lack a thick protective cell wall which prevents the direct contact of nanoparticles with cytoplasm. Therefore we carefully studied the effects of magnetic functionalization of HeLa cells with PAH-stabilized SPIONs. We have chosen three major factors, namely, membrane integrity, enzymatic activity, and ability to grow and colonize substrates as indicators of cells’ viability. First, we applied the LIVE/DEAD kit consisting of a mixture of SYTO10 highly permeable dye staining DNA in all cells (including those with intact membranes) and cell impermeant DEAD Red dye, which stains nucleic acids only in cells with compromised membranes.17 The simultanous use of those dyes allows us to illustrate the ratio of living (green) and dead (red) cells in a mixture via the selective binding of fluorescent dyes to cellular DNA. As shown in Figure 5A, the majority of SPION-coated cells were viable, indicating no significant damage to cellular membranes. Further, to investigate the influence of SPIONs on intracellular enzymes, we employed the FDA test.18 Nonfluorescent FDA travels through the
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Figure 5. Viability of SPION-functionalized HeLa cells. Fluorescence microscopy images of (A) of LIVE/DEAD stained and (B) FDA stained SPION-functionalized HeLa cells. (C) Flow cytometry data demonstrating the viability of FDA-stained SPION-functionalized HeLa cells.
membrane, where it undergoes an enzymatic decomposition and stains only the living cells, whereas in dead cells inactive esterases and damaged membranes prevent the accumulation of the fluorescent dye inside the cytomplasm (Figure 5B). We applied flow cytometry to estimate the effect of the SPIONs on viability quantitatevely. As one can see in Figure 5C, 83% of cells were viable, which is close to the number of viable cells in control samples (88%). Furthermore, to show that the SPION-coated cells not only remain viable, but also are able to grow and colonize suitable substrates, we introduced SPION-functionalized cells (ca. 3 105 cells/mL) into cell culture plates containing the growth media and incubated them at 37 °C, using intact cells as a control. As shown in Figure 6, the magnetized cells colonized the substrate as well as the intact cells, reaching 75% confluency after 5 days of incubation. This also suggests that PAHstabilized SPIONs form a flexible coating on the cells, which does 14390
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Figure 6. SPION-functionalized cells are able to grow on substrates: optical microscopy images of (A, C) intact HeLa cells and (B, D) SPIONfunctionalized HeLa cells after 2 and 5 days growth in culture plates filled with DMEM.
Figure 7. Magnetically facilitated microorganization of SPION-functionalized HeLa cells. (A) A sketch demonstrating the position of 2 mm NeFeB magnets below the culture well. Photographs of culture wells used to harvest (B) intact and (C) SPION-functionalized HeLa cells. Optical microscopy images of (D) intact and (E, F) SPION-functionalized HeLa cells microorganized around the permanent magnet (note the spindle-shaped cells attached to the substrate).
not prevent the settling of the cells and the formation of lobopodia, exactly as it happens in intact cells.22 In a set of separate experiments, we added the SPIONs over the intact cells already placed in the wells and found that they cause no alteration in the growth of the cells (data not shown). Therefore, we
conclude that PAH-stabilized SPIONs are nontoxic to HeLa cells, which we attribute to the previously reported low toxicity of both iron oxide nanoparticles23,24 and PAH.25 In addition, the straightforward approach described here does not require sequential deposition of polymer layers doped with nanoparticles 14391
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’ CONCLUSION We believe that the approach reported here opens new avenues for the selective positioning of SPION-functionalized cells. In this paper we demonstrate for the first time that the isolated human cells can be functionalized via a single-step facile process using PAH-stabilized biocompatible SPIONs. The novel strategy realized here is based on electrostatic deposition of biocompatible polymer-stabilized SPIONs on the surfaces of the cells, where no specific ligand receptor interaction31,32 is required; hence, this technique may be applied to other human cells as well. The cells preserve their viability after the modification. The SPION-functionalized cells can be manipulated using an external magnetic field, which allows their spatial accumulation at certain desired points and further growth at those points. We suppose that the route shown here using HeLa cells is
applicable to other types of isolated human cells, thus potentially making it an important instrument in cell-based therapies. In addition, the controllable magnetically facilitated deposition of SPION-functionalized human cells may find applications in development ofwhole-cells biosensors.
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional figures as mentioned in the text and real-time footage of magnetically facilitated movement of functionalized cells. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Tel: +78432337833. Present Addresses
cole Laboratoire de Neurobiologie, Departement de Biologie, E Normale Superieure, 46 rue d’Ulm, 75005, Paris, France.
)
onto the cells, as reported earlier,25 which also may reduce the possible toxic effects. Finally, we show here that the SPION-coated HeLa cells can be spatially organized in multicellular living clusters using an external magnetic field. We placed 2 mm cylindrical permanent magnets below the cell culture plate wells (as shown in Figure 7A), which contained 2 mL of growth media and then added 1 mL of SPION-functionalized HeLa cells (3 105 cells mL 1) in DPBS. In control samples we used intact HeLa cells. The culture plates were incubated at 37 °C. After 2 days we observed the formation of visible multicellular clusters right above the position of the magnets (as shown in Figure 7C), whereas no spatial organization was observed in control samples (Figure 7B). The cells in control samples were positioned randomly; therefore, we assume that SPION-functionalized cells travel to the magnets and then attach to the substrate and grow around the magnets. Microscopic observations confirmed that the cellular clusters repeat the geometry of the magnet while concentrating in round-shaped formations (Figure 7E); at the same time the intact cells randomly colonize the whole area of the well (Figure 7D). The characteristic spindlelike shapes of growing cells can be clearly seen on the edges of the round-shaped clusters (Figure 7F), indicating that the cells actively divide and grow at the certain area outlined with a permanent magnet. Several recent papers suggest various techniques aimed to functionalize microbial and human cells with polyelectrolytes, nanoparticles, or mineral coatings.26 30 Magnet-facilitated spatial distribution on the culture plates and the subsequent growth of the SPION-functionalized cells indicates that this technique may be potentially extended to controlled deposition and magnetic-field-oriented growth of functionalized cells in more complex systems. On the basis of our results reported in this paper, we believe that biocompatible cell-adherent SPIONs may be successfully deposited onto other types of isolated human cells, including stem cells, and then utilized in tissue engineering and cell-based therapies. For the former case, SPION-functionalized cells can be assembled using an external magnetic field in complex patterns, followed by the growth and tissue formation, whereas for the latter case, SPION-functionalized cells may be delivered into the certain region of the body. Obviously, a great deal of further research must be performed prior to application of SPION-functionalized human cells in humans; however, our current results, as we believe, will inspire scientists to apply the simple and direct technique reported here in tissue engineering and cell-based therapies.
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Author Contributions §
These authors contributed equally.
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