RAPID CELLULAR UPTAKE OF SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES BY USING LOW-INTENSITY ULTRASOUND

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1 RAPID CELLULAR UPTAKE OF SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES BY USING LOW-INTENSITY ULTRASOUND Mary KOLÁŘOVÁ, Kateřina POLÁKOVÁ, Kateřina TOMÁNKOVÁ, Markéta HAVRDOVÁ, Zdenka MARKOVÁ, Radek ZBOŘIL Palacky University Olomouc, Olomouc, Czech Republic, EU Abstract Nowadays, in cell therapies in-vivo monitoring of target cells has become highly desirable. For this purpose SPIO (Superparamagnetic Iron Oxide) nanoparticles are suitable as magnetic detectable markers of the transplanted cells by using non-invasive magnetic resonance imaging (MRI). To improve the labeling efficiency of cells by SPIO nanoparticles various methods such as using transfection agents or electroporation have been used, however with negative impact on cell viability. In this study we introduce a low-intensity therapeutic ultrasound as a safe and non-invasive method for cell labeling with SPIO nanoparticles. The human fibroblasts alone and with addition of SPIO nanoparticles were treated by commercial low-intensity ultrasound (BTL 4000, USA). By changing of starting parameters such as intensity (from 0.1 to 2 W/cm 2 ), exposure time (1, 3 and 5 min) and various SPIO concentrations we have found the optimal labeling protocol having minimal impact on cell viability and maximal MRI contrast effect. The cellular uptake of SPIO nanoparticles was detected by Prussion blue staining and by atomic absorption spectroscopy (AAS). The viability of cells was determined by using MTT test. Contrast effect of labeled cells was confirmed by clinical 1.5 T MRI. Keywords: superparamagnetic iron oxide nanoparticles (SPIO), fibroblasts, sonoporation, MRI, cell labeling 1. INTRODUCTION Ultrasound also known as sonography has become widely used as a diagnostic, surgical and therapeutic tool in clinical practice [1]. Another possible utilization of sonography is a labelling of cells by SPIO nanoparticles or transfection of drugs or genes into the cells using sonoporation. In this case the ultrasound transmits ultrasonic waves causing temporary changes of permeability in the cell membrane followed by creating membrane pores. Nanoparticles or drugs are thus easily transported through these pores from extracellular to intracellular area by physical effect without any chemicals. SPIO nanoparticles with diameter up to 100 nm are usually occurred in the form of Fe3O4 called magnetite or γ-fe2o3 called maghemite [2]. For bioapplications SPIO nanoparticles should be stabilized by biopolymer layer of dextran, carboxydextran, starch, albumin, silicone and polyethylene glycol to prevent their aggregation and enabling further ligand conjugation [3]. MRI imaging is a non-invasive diagnostic method enabling visualization also soft tissues. To enhance resolution and contrast of the images usage of appropriate contrast agents is required [4]. Contrast agents based on SPIO nanoparticles shorten T1 and T2 relaxation times of labelled tissue due to the fast proton relaxation. Therefore, we are able to observe contrast changes at the cellular level [5]. There are several commercial contrast agents based on SPIO nanoparticles depending on further application (intravenous or oral) [5]. Cellular magnetic resonance imaging is a useful approach for in-vivo tracking of cells transplanted for therapeutic purposes. Magnetic cell labeling, which involves incorporating of magnetic nanoparticles into the intracellular space of the cells, is an essential step for this technique [6]. In the study of Mo et al. (2010) it was observed that cell labelling with SPIO nanoparticles by using ultrasonic waves has increased efficiency of labelling.

2 In this study we have focused on the labelling of fibroblasts cells by our new SPIO nanoparticles using low field therapeutic ultrasound to find out the optimal labelling protocol for future in-vitro and in-vivo studies. 2.1 Cells culture Fibroblast cell line was grown under the standard conditions in a nutrient medium with fetal bovine serum and antibiotics (penicillin, streptomycin) in a thermostat at 37 C, humidity 95 % and 5 % CO2. The cells adhered on the well plates were always passaged just before the experiment was started. First of all, the medium was drained out and washed twice with a phosphate buffered saline (PBS) 37 C. Then trypsin EDTA solution was added in order to release the cells from the bottom of the well plates. After that it is necessary to check the cells by microscope in order to avoid losses since the cells in each well plate deadhere in a different time. The well plates were washed with PBS and all cells were put into the test tube of 10 mm diameter and 50 mm length. Test tube with cell suspension was centrifugated at 1500 rpm for 4 min in a room temperature. The sample was washed by new medium and then filled up to 1 ml. The test tube with cell suspension was placed in a water bath. 2.1 Superparamagnetic iron oxides nanoparticles Superparamagnetic nanoparticles smam were prepared in laboratory in Regional Centre of Advanced Technologies and Materials (RCPTM). These nanoparticles, maghemite (γ-fe2o3), were prepared from a precursor of ferrous sulphate (FeSO4) by thermally induced reactions in the aqueous phase in the presence of the polymer poly(acrylic-co-maleic) acid. This polymer prevents aggregation of magnetic particles during nucleation and also avoids a crystal growth during the synthesis. It has also free functional carboxyl groups with a negative charge. The mean particle size was determined as 20 nm. The particles forms regular agglomerates with flower shape with a size of about nm. 2.3 Ultrasound exposure For the experiment we used a therapeutic ultrasound (BTL 4000, USA). The ultrasound apparatus transmits a radiofrequency sinusoidal signal with appropriate intensity, frequency and exposure time. Frequency of ultrasound is adjustable to 1 MHz and 3 MHz. The maximum intensity is 3 W/cm 2 in a pulsed mode and 2 W/cm 2 in a continuous mode. Ultrasound exposure scheme and its system are illustrated in Fig. 1. The ultrasound exposure system consists of a transparent container filled with water up to its upper edge, then of a head for heating this water and a holder for test tube on the top. Waterproof ergonomically shaped ultrasonic probe is placed in the water bath at 1/3 height of the apparatus. The distance of central focal spot is 7.2 cm from the sound source. Fig. 1 Scheme of the experimental setup for the low-intensity pulsed ultrasound labeling of cell culture Fibroblast cells were prepared according to paragraph 2.1. SPIO nanoparticles with various concentrations were applied into the each test tube containing cell suspension. There were seven groups in this study: A test tube with no SPIO nanoparticles; Ba SPIO nanoparticles smam with concentration 250 μg/ml exposed

3 for 1 min; Bb smam 250 μg/ml, 3 min; Bc smam 250 μg/ml, 5 min; Ca smam 500 μg/ml, 1 min; Cb smam 500 μg/ml, 3 min; Cc smam 500 μg/ml, 5 min. The samples in the test tube were mixed and then fixed in a water bath. The fibroblast cells were treated by intensity 0.1 W/cm 2. After ultrasound exposure of the each sample (Ba - Cc) the cells were centrifugated. The supernatant was poured out and pellet was washed properly with PBS. This helps to remove all nanoparticles that are only adhered on the cell surface or nanoparticles founded just around the pellet in supernatant. Afterwards, the samples were centrifugated again at 6 C. This process was repeated four times to provide a pellet containing only those cells having SPIO nanoparticles inside the intracellular space (Fig. 2). The efficiency of labelling process using low-intensity ultrasound was compared with a standard incubation. The process of incubation has been described elsewhere [6]. Fig. 2 Samples with a visible pellet after the second, third and fourth centrifugation. A C: pellet of cells with no smam nanoparticles (black arrows). D F: pellet of cells contains smam nanoparticles (black arrows) and the gradual removing of non-incorporated smam nanoparticles (white arrows) 2.4 Analysis of labeling efficiency with Prussian blue staining and in vitro MRI experiment Cells were washed and harvested by centrifugation after sonoporation. They were seeded back into well plate. The cells were fixed with Prussian blue staining (Fe4[Fe(CN6)]3) and incubated for 10 min to detect the iron from SPIO. The blue dye was quantified using an optical microscope. For MRI measurement a range of concentrations input of SPIO nanoparticles labeled by sonoporation was setting up: c1= 35 μg Fe/ml, c2 = 100 μg Fe/ml, c3 =180 μg Fe/ml, c4 = 250 μg Fe/ml, c5 = 500 μg Fe/ml, c6 = 1000 μg Fe/ml, c7 = 0 μg Fe/ml. Subsequently, phantoms for MRI imaging were prepared from these labeled cells mixing in 1 % agar. The negative contrast effect of cells was checked by clinical 1.5T MRI tomography. 2.5 Measurement of iron content Atomic absorption spectroscopy (AAS) is a method based on absorption of optical radiation by free atoms in the gaseous state. This method allows determination of iron concentration in the cell sample. After sonoporation and centrifugation samples of cells labeled with SPIO nanoparticles were boiled within HNO3 at temperature 100 C for 20 min. After that samples were diluted with distilled water up to 5 ml and measured by atomic absorption spectroscopy to obtain the final amount of iron in each sample. 2. RESULTS AND DISCUSSION The cellular uptake of iron oxide nanoparticles was assessed visually using an optical microscope and detected by Prussion blue staining. The labelling efficiency was detected as a rate of blue stained cells to the

4 total number of cells. We also monitored morphological changes of fibroblasts after Prussian blue staining. The shape of the cells becomes rounded (Fig. 3b). A possible explanation for this effect is that cells are imaged in the time when they are not fully adhered on the surface of well plate. More important is the fact that blue coloured part of the cell body demonstrates a presence of iron oxide nanoparticle inside the cells. Figure 3 depicts nanoparticles occurred in the cells. In the case of fibroblasts after sonoporation process SPIO are located only in the peripheral part of the cell body in contrast to standard incubation where nanoparticles are centralized close to the cell nucleus of stem cells (Fig. 3a). To gain a quantitative amount of the iron observed in the sample an atomic absorption spectroscopy has been used. Thus, after recalculating, we can get the information about the amount of the iron per one cell. Fig. 3 Optical images of cells after Prussian blue staining (A) Stem cells labeled with SPIO nanoparticles by standard incubation, (B) fibroblasts labeled by sonoporation. Black arrows indicate the SPIO nanoparticles on the surface of cells, red arrows indicate nanoparticles incorporated in the cells We supposed that the amount of iron per cell is going to increase with exposure time of sonoporation during the labelling process. However, the biggest uptake of SPIO nanoparticles occurred within the first minute. With used concentration of 250 µg Fe/ml and exposure time 1, 3 and 5 min we reached 4.10 pg Fe, 27.5 pg Fe and 2.56 pg Fe per one cell. Higher uptake of iron oxide nanoparticles was found at concentration of 500 µg Fe/m 8.46 pg Fe, 6.3 pg Fe and 5.8 pg Fe per one cell by using the 1, 3 and 5 min of sonoporation (see Figure 4). These results show that the efficiency of labeling of cells by sonoporation is inversely proportional to the exposure time. However, an increasing input concentration of nanoparticles increases also the cellular uptake. The highest uptake of nanoparticles into the cell, /- 2.8 pg Fe per one cell, has been reached by application of 500 μg Fe/ml and exposure time 1 min. These values are comparable to the results of Runyang Mo et al. (2011) [7]. They observed a value of / pg Fe per cell with initial nanoparticles concentration of 410 µg Fe/ml. Some studies have even stated an uptake above 60 pg of iron per cell. But the results strongly depend on the type of used cell line, nanoparticles and also setting of ultrasound apparatus. For comparison, a control sample of unlabeled cells contains nearly 0 pg of iron per cell. As was mentioned above, 5 minute sonoporation caused a decrease of labelling efficiency. Runyang Mo at al. (2011) [7] indicates as a possible explanation that the acoustic cavitation in combination with floating nanoparticles can cause apoptosis. Long exposure time could also lead to transcytosis. Due to a flowing of water nanoparticles could be through the membrane pores also ejected out of the cells. Also saturation of amount of incorporated SPIO nanoparticles in the first minute could be a reason for the highest labelling efficiency in the first minute. Dependence of used exposure times on cell labelling is studied by Yi-Xiang J. Wang at al. (2010) [8]. Contrary to our results, the cellular uptake increases linearly with increasing exposure time [8]. But they used different parameters of ultrasound and also different SPIO nanoparaticles.

5 Fig. 4 The dependence of concentration of iron per cell on the exposure time determined by atomic absorption spectroscopy The results of labeling efficiency (the amount of iron per cell) by sonoporation were compared to standard incubation using atomic absorption spectroscopy. Table 5 shows the value 8,46 pg per cell in case of labeling by sonoporation. It is a slightly higher value then the amount of iron obtained during incubation (6.76 pg per cell). These results are in a good agreement with a study of Yi-Xiang J.Wang at al. (2010) [8] where they also found a significant advanced effect of labelling of tumour cells (osteosarcomas) by using ultrasound. Figure 5 Comparison of the concentration of iron per one cell labeled by sonoporation (1 min) and standard incubation (24 h) Magnetic resonance imaging (MRI) with its high resolution and contrast properties provides a monitoring of cells labelled with SPIO nanoparticles. We have prepared a series of phantom samples differing in input SPIO concentration before sonoporation process. The samples c1 to c7 with concentrations of 35 to 1000 µg Fe/ml have the values of negative contrast as 741, 400, 391, 210, 174, 125, 1164 (agar) established from MRI software. Figure 6 demonstrates the images of phantoms containing magnetically labelled cells with c1 to c7 SPIO concentrations. Phantom no.6 has initial concentration 1000 μg Fe/ml and achieves the biggest negative signal. It is therefore confirmed that with increasing concentration of iron oxide nanoparticles in sonoporation process increases also the content of iron per cell which mean the better MRI contrast properties of the labelled cells. This fact was also confirmed by atomic absorption spectroscopy and by Prussian blue staining.

6 Figure 6 Negative contrast effect of cell samples labelled with various SPIO concentrations; Notice: Number 7 is a blind agar sample 3. CONCLUSION The purpose of this study was to optimize the cell labelling protocol with superparamagnetic iron oxide nanoparticles. The final parameters of the ultrasound, by which the highest value of iron (8,46 +/- 2,8 pg Fe per one cell) was found, were achieved as 0.1 W/cm 2, 1 MHz, 1 min of exposure time and 500 μg Fe/ml of initial SPIO concentration. These results suggest that the low intensity ultrasound, after optimization of input parameters, can be used as a highly effective and non-toxic technique for labelling of cells by SPIO nanoparticles. ACKNOWLEDGEMENT Financial support by the European Regional Development Funds (CZ.1.05/2.1.00/ ) and KONTAKT II LH LITERATURE [1] BOHARI, S.P., GROVER, L.M., HUKINS, D.W. Pulsed-low intensity ultrasound enhances extracellular matrix production by fibroblasts encapsulated in alginate. Journal of Tissue Engineering, [2] VARADAN, V.K., Chen, L., X.J. Design and applications of magnetic nanomaterials, nanosensors and nanosystems. Nanomedicine. John Wiley & Sons, [3] REDDY, A.M. et al. In vivo Tracking of Mesenchymal Stem Cells Labeled with a Novel Chitosan-coated Superparamagnetic Iron Oxide Nanoparticles using 3.0 T MRI. Journal of Korean Medical Science, , [4] Liu, Z. et al. Advanced nanomaterials in multimodal imaging: design, functionalization, and biomedical applications. Journal of Nanomaterials, 2010, [5] SYKOVÁ, E., JENDELOVÁ, P. Magnetic Resonance Tracking of Implanted Adult and Embryonic Stem Cells in Injured Brain and Spinal Cord. Annals of the New York Academy of Sciences, 2005, 1049, [6] CHIAO-CHI, V.CH. Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI. PLOS ONE, 2013, 8, [7] MO, R. et al. Instant magnetic labeling of tumor cells by ultrasound in vitro. Journal of Magnetism and Magnetic Materials, 2011, 323, [8] WANG, Y.X. et al. Low-intensity pulsed ultrasound increases cellular uptake of superparamagnetic iron oxide nanomaterial: results from human osteosarcoma cell line U2OS. Journal of Magnetic Resonance Imaging, 2010, 31,