Biodistribution of poly clustered superparamagnetic iron oxide nanoparticle labeled mesenchymal stem cells in aminoglycoside induced ototoxic mouse model

Graphic abstract

Recently, application of stem cell therapy in regenerative medicine has become an active field of study. Mesenchymal stem cells (MSCs) are known to have a strong ability for homing. MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIONs) exhibit enhanced homing due to magnetic attraction. We have designed a SPION that has a cluster core of iron oxide-based nanoparticles coated with PLGA-Cy5.5. We optimized the nanoparticles for internalization to enable the transport of PCS nanoparticles through endocytosis into MSCs. The migration of magnetized MSCs with SPION by static magnets was seen in vitro. The auditory hair cells do not regenerate once damaged, ototoxic mouse model was generated by administration of kanamycin and furosemide. SPION labeled MSC’s were administered through different injection routes in the ototoxic animal model. As result, the intratympanic administration group with magnet had the highest number of cells in the brain followed by the liver, cochlea, and kidney as compared to those in the control groups. The synthesized PCS (poly clustered superparamagnetic iron oxide) nanoparticles, together with MSCs, by magnetic attraction, could synergistically enhance stem cell delivery.

The poly clustered superparamagnetic iron oxide nanoparticle labeled in the mesenchymal stem cells have increased the efficacy of homing of the MSC’s to the target area by synergetic effect of magnetic attraction and chemotaxis (SDF-1/CXCR4 axis). This technique allows delivery of the stem cells to the areas with limited vasculatures. The nanoparticle in the biomedicine allows drug delivery, thus, the combination of nanomedicince together with the regenerative medicine will provide highly effective therapy.

Introduction

Mesenchymal stem cells (MSCs) possess great potential as therapeutic agents [1, 2]. MSCs has the ability to differentiate into several lineages of mesenchymal origin such as fat, cartilage, bone, tendon, and muscle [3]. Although MSCs can be applied in various clinical settings, limitations associated with their delivery to the target site or injury area remains an obstacle. The delivery of an appropriate number of cells to defective tissue remains difficult. Devine et al. demonstrated a low engraftment efficacy, estimated to range from 0.1 to 2.7%, in the kidney, lung, liver, thymus, and skin [4]. Recently, various strategies have been developed to improve MSC homing and engraftment. The mechanisms involving MSC homing has been demonstrated as tethering, rolling, adhesion, and transmigration into the injured site [5]. Strategies such as increasing the biological effects of lipid rafts, modifying the expression and function of homing molecules (CXCR4/SDF-1 axis), modifying the metabolism of MSCs, or enhancing the availability of chemotactic factors for MSCs have been applied [6–8]. Although engineered MSCs express certain cell-surface receptors, including some chemokine receptors, that mediate various aspects of homing, they do not express all essential chemokines and have low expression levels of other important adhesion receptors (e.g., CXCR4), which regulate the tethering and rolling of circulating cells on the activated vascular endothelium [9].

Technology involving nanoparticles can be used for magnetic attraction and cellular tracking. Nanoparticles internalized in the stem cell could enhance the homing by applying magnetic attraction. Superparamagnetic iron oxide nanoparticles (SPIONs) have been developed for monitoring the migration of injected stem cells by magnetic resonance imaging (MRI). Shen et al. showed a promising technique to deliver stem cells by increasing homing and retention of magnetically labeled MSCs in traumatic brain injury by applying a static magnetic field [10]. In our previous study, we applied static magnets to control SPION labeled stem cells, in the olfactory injury mice model [2]. Although the SPION is widely used in biomedical applications, modifications in SPION concentration, size, surface charge, would lead oxidative stress which will exert cytotoxic effects. Further research on nanoparticles and verifying clinical safety measures will make stem cell homing clinically viable.

Aminoglycoside antibiotics have been widely used against bacterial infections, such as tuberculosis [11]. However, they can cause cochleotoxicity and vestibulotoxicity, leading to hearing loss. Stem cell therapy is essential in this case because auditory organs, once damaged, tend to not regenerate. The ear has a complex microstructure, with several tiny capillaries, making it difficult for stem cells to home into the hair cells of the cochlea. In addition, the delivery methods, like intravenous or intratympanic injection, of stem cells to the cochlea, are not well studied [12]. In this study, we demonstrated the homing of nanoparticle labeled stem cells to the cochlea and their distribution, in response to delivery route. An ototoxic animal model was generated by administering a combination of aminoglycosides and loop diuretics to induce complete hearing loss. We have synthesized poly clustered superparamagnetic iron oxide (PCS) nanoparticles by using Poly(d,l-lactic-co-glycolic acid) as the shell and iron oxide magnetic nanoparticle as the core; properties of the nanoparticle and its stability were also tested. In vitro experiments indicated that the PCS nanoparticles did not change the viability and characteristics, such as migration ability, differentiation ability, and proliferation, of the MSCs. Our study showed that the PCS nanoparticles were suitable for in vitro and in vivo experiments; in the presence of an external magnet they could induce stem cell migration, thus improving the homing capacity of the MSC, and increasing their efficacy in clinical use.

Materials and methods

Synthesis of PCS nanoparticles

PCS nanoparticles were synthesized by using the following: poly(D,L-lactide-co-glycolide) (50:50, MW 38,000–54,000) (PLGA, Sigma-Aldrich, USA), iron oxide (II, III), magnetic nanoparticles solution (SPION), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo-fisher scientific, Massachusetts, USA). N-hydroxysuccinimide (NHS) was used as a linker between the fluorescent molecule. Cy5.5 (Cyanine 5.5 amine, red; Lumiprobe Co., Hannover, Germany) was synthesized for detecting nanoparticles in the cells.

PCS nanoparticles, termed as PLGA-Cy5.5, were synthesized by EDC-NHS coupling; 330 mg of PLGA was dissolved in DMSO, with 60 mg of EDC, 132 mg of NHS, and 9.9 mg of Cy5.5, and incubated for 24 h. The nanoparticle mixture was extensively dialyzed (MW cutoff = 10 k) with deionized water to remove excess EDC, NHS, and Cy5.5. The resulting PLGA-Cy5.5 particles were freeze dried to obtain powdered samples.

Characterization of PCS nanoparticles

PCS nanoparticles, with the polymeric shell consisting of 10 nm sized SPIONs in the core, were prepared using an oil-in-water (O/W) emulsion method. A total of 1 mg of PLGA-Cy5.5 was dissolved in acetonitrile at 25 °C and to this solution 0.1 mL of SPION (5 mg/mL) solution was added drop wise in 3 mL of deionized water. The vial was vortexed for 5 min, followed by 3 min of sonication. The mixture was stirred at room temperature for 6 h. The nanoparticles were further purified using an ultra-centrifuge and stored at 4 °C until further use.

The size and surface zeta potential of the nanoparticles were obtained by dynamic light scattering (DLS) (Malvern, Zetasizer-ZS90). The morphology of the particles was characterized using a scanning electron microscope (SEM) (JEOL, JSM-7100F). SEM samples were prepared by adding 2 µL of nanoparticle suspension onto a polished silicon wafer. Droplets were desiccated at room temperature for 4 h; the sample was coated with platinum and imaged using SEM. TEM measurement was performed to analyze the SPION core. Samples for TEM imaging were prepared by the drop casting over a carbon grid.

Hedayati’s method [13] was applied to quantify the Fe ion concentration of the PCS nanoparticle solution. Briefly, for measuring the concentration of the Fe (iron) ion, acetate buffer, ferene-s solution, working buffer, and working solution were made. A total of 7.7 g of ammonium acetate (Fisher cat. No. A637-500, FW = 77.08) was added to 20 mL of glacial acetic acid (Fisher cat. No. A38-212) and mixed with DI water, to a final volume of 50 mL, to prepare the acetate buffer. The ferene-s solution was prepared by adding 0.5 g of ferene-s (Sigma cat. No. P4272, FW = 494.37) to 2 mL of DI water. To make the working buffer, 400 mg of l-ascorbic acid (Sigma cat. No. A92902-100, FW = 96.12) was added to 2.2 mL of acetate buffer. Finally, 2 mL of working buffer, 0.1 mL of ferene-s solution, and 7.9 mL of DI water were mixed to make the working solution. A total of 50 µL of PCS nanoparticle solution was mixed with 950 µL of working solution.

In vitro study

Cell culture and labeling

Mouse MSCs tagged with green fluorescent protein were cultured under 37 °C and 5% CO₂ in complete culture medium with MEM (Hyclone, UT, USA), supplemented with 10% FBS (Gibco, NY, USA), 1% penicillin/streptomycin, and amphotericin B. Labeling of the MSCs with the nanoparticles was performed with laboratory synthesized, 100 nm polymeric clustered superparamagnetic iron oxide nanoparticles (PCS nanoparticles). Adherent cells were incubated with 40 µg/mL PCS nanoparticle suspended media for 24 h.

Cell viability assessment

Cell proliferation and viability were quantified by using EZ-cytox cell viability assay kit (Daeil Lab., Seoul, Korea). Cells were seeded in a 96-well plate and were labeled with PCS nanoparticles in concentrations of 10, 20, and 40 µg/mL and absorbance was measured with a spectrophotometer, at 450 nm.

Migration assay

Migration assays were performed by using Costar migration chambers (Transwell® 6.5 mm diameter chambers, 8.0 µm pore size, 24-well, Kennebunk ME, USA). PCS nanoparticle labeled MSCs were plated at a concentration of 2 × 10⁴ in the upper chamber of the well, supplemented with media containing 1% FBS. Complete culture media with the chemoattractant, SDF-1, was placed in the lower chamber for 24 h, and 5 mm cubic neodymium were placed at the bottom of the culture plate to observe the migration of MSC with or without influence of magnetic field. The membranes were fixed with PFA and stained with crystal violet and observed under the light microscope.

Immunofluorescence

Green fluorescent protein tagged MSCs were seeded in a 1-well culture dish and the cells were left to attach; 40 µg/mL of nanoparticles were added in the culture medium to label the cells for 24 h. After labeling, the cells were PBS washed at least twice and the culture media was added. A 3 mm NdFeB magnet, with magnetic force of 0.34 T, was attached at one side of the culture slide and the cells were magnetically attracted for 24 h. Magnetically attracted cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS twice. The nuclei were counterstained with DAPI (Fluoroshield™ with DAPI, Sigma-Aldrich, Co., MO, USA), a coverslip was placed on the samples, which were then left to dry for 24 h. The samples were observed under the fluorescence microscope. Fluorescence images were taken from the culture slide.

In vivo study

In vivo experiments were performed in the animal laboratory of Yonsei University Wonju College of Medicine Wonju, Korea in accordance with the policies of the institutional animal care and use committee (YWC-190513-1).

Animal and test group

C57BL/6 mice, (males, 5–6 weeks old, weighing 18–25 g) were used for the experiment. The animals were maintained at room temperature and had free access to food and water. Fifteen mice were individually caged and grouped into test group 1 (MSC injection via IV injection; n = 5), test group 2 (MSC administration via IT injection with magnet application; n = 5), and test group 3 (MSC administration via IT injection without magnet application; n = 5).

Ototoxic animal model and abr recording

The ototoxic drugs, kanamycin sulfate 550 mg/kg (VWR life sciences, PA, USA) and furosemide 130 mg/kg (Lasix, Handok, Korea), were given through subcutaneous and intraperitoneal injection. Animals were anesthetized with 100 mg/kg ketamine (Yuhan, Seoul, Korea) and 10 mg/kg xylazine (Rompun, Bayer, Ansan, Korea) by intraperitoneal injection before ABR recordings. The mice were tested in a sound attenuating chamber with a built-in Faraday cage; an isothermal pad was used to maintain the body temperature. For stimulus generation, data management, and ABR collection, the TDT RZ6/BioSigRZ system (Tucker Davis Technologies, Alachua, FL, USA) was used. Subdermal electrodes were placed to collect data from the mouse; the reference electrode, which was on the same side as the stimulus, was placed axial to the pinnae; the ground electrode was placed in the ipsilateral ear; and the active electrodes were placed in the vertex. ABR was recorded 1 day prior to administration of the ototoxic drug and 7 days post administration of the ototoxic drug.

PCS nanoparticle labeling

Poly(lactic-co-glycolic acid) coated polymeric clustered superparamagnetic nanoparticles utilized in the experiments were synthesized in the laboratory. Nanoparticles were added to the cell culture media and incubated together with the attached cells for 24 h. After labeling, the cells were washed with PBS at least twice, suspended in PBS and counted according to the administration density.

PCS nanoparticle labeled MSC transplantation

Nanoparticle labeled stem cells were administered through different injection routes. Prior to administration of the nanoparticle labeled stem cells, the animals were anesthetized with 100 mg/kg ketamine (Yuhan, Seoul, Korea) and 10 mg/kg xylazine (Rompun, Bayer, Ansan, Korea) by intraperitoneal injection. For systemic transplantation, the cells were introduced slowly through the tail vein by a 31-G needle. To transplant the cells directly into the target area, intratympanic administration were performed by infusing the cells into the tympanic cavity with 31-G needle capped with polyimide tubing (Cole-palmer®, IL, USA); cell overflow or leakage was prevented by placing the subject lying contralateral to the infusion site. Transplantation events required 40–50 s each.

Magnet application

Incision was made in the skin around the vertex of the skull and 5 mm cuboidal NdFeB (neodymium iron boron) magnet transplanted subcutaneously and was sutured post administration of the nanoparticle labeled stem cells.

Fluorescence imaging of tissue samples

Tissue samples from different organs, 24 h post MSC administration, were collected and fixed in 4% paraformaldehyde for 48 h. Tissue samples were then embedded in paraffin and cut into 5 µm-thick-sections with a microtome. To observe the green fluorescence protein (GFP) emitting MSCs, the sections were deparaffinized and the nuclei were counterstained with DAPI (Fluoroshield™ with DAPI, Sigma-Aldrich, Co., MO, USA). The distribution of the GFP emitting cells in the tissue sections was measured with the ImageJ software.

Reverse transcriptase PCR

To quantify the nanoparticle labeled cells in different tissue samples, total RNA from the liver, kidney, brain, lungs, heart, and cochlea was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). A total of 1 µg of RNA was reverse transcribed using ReverTra Ace® qPCR RT master mix with gDNA remover (Toyobo, Osaka, Japan) and cDNA was used as a template for PCR amplification. Reverse transcriptase PCR was performed using Applied Biosystems SimpliAMP thermal cycler to quantify the GFP levels. The following primers were used: GFP, forward: 5′-ACT TCA AGA TCC CGC ACA ACA T-3′ and reverse: 5′-TTA CTT GTA CAG CTC GTC CAT CG-3′. Duplicate measurements were performed and the result was accepted only if the difference in the Ct values was less than 1.

Fluorescence imaging of organs ex vivo

The mice were euthanized 24 h post MSC injection and the organs were removed. All animal experiments were performed with the approval from the Institutional Animal Care and Use Committee of Yonsei University (IACUC, YWC-190513-1, May 2019). The intensity of GFP fluorescence was measured by FOBI (Neoscience, Suwon, Korea). GFP intensity of was normalized for validating the biodistribution of MSCs. The equation of normalized mean fluorescence intensity (MFI) is as follows.

$$\text{Normalized organ fluorescence}=\frac{\text{Organ fluorescence}}{\text{Sum of organ fluorescence}}\times 100\left(\%\right)$$

Results

Characterization of PCS nanoparticles

Nano-sized PCS nanoparticles (PSCs) were prepared by the bottom-up method, in oil-in-water emulsion, as described previously [2]. Schematic representation of PCS nanoparticle preparation is showed in Fig. 1a. PCSs have two components, (1) an oleic acid-coated SPION core, which confers high stability to the nanoparticle, and (2) a PLGA-Cy5.5 polymer shell, which enables the nanoparticle to interact with the aqueous phase and increases the biocompatibility of the nanoparticle with the fluorescent moiety.

Fig. 3.

MSCs time-dependent migration to the magnet in vitro (3 mm) 24 h and 48 h. a Fluorescent microscopic images of magnetic attraction test in vitro at 24 h (up) and 48 h (down). b Quantification of magnetic attraction test at 24 h (left) and 48 h (right) (*p < 0.05, n = 3)

The physicochemical properties of PCSs were measured by Zetasizer-ZS90. The hydrodynamic diameter was calculated to be 114 nm, with 0.162 poly-dispersity index (PDI), using dynamic light scattering (DLS) (Fig. 1b). The surface ζ-potential was − 29.6 mV, under aqueous conditions, which means that the carboxyl group in PLGA was on the nanoparticle surface (Fig. 1c). The SEM images showed that the PCS nanoparticles were spherical and had a polymer shell (Fig. 1d). TEM showed that the SPION core was present in the particles (Fig. 1e). Thus, the PCS nanoparticles were approximately 100 nm in spherical diameter, with a negative surface ζ-potential. The PLGA-Cy5.5 shell provides high biocompatibility and biodegradability. The SPION core provides the PCS nanoparticles magnetization property [3]; an external magnet can guide the PCS nanoparticles to the target sites [4]. The ferene-s assay, to measure Fe ion, was accurate in the range of 0–4 µg/mL of Fe ion concentration (Fig. 1f). To validate the synthesis of PLGA-Cy5.5, Fourier-transform infrared spectroscopy (FT-IR) was used (Fig. 1g). Compared to the PLGA peak, the amide I peak was detected at 1626 cm^− 1, and the amide A peak was detected at 3324 cm-¹ in the PLGA-Cy5.5 peak. These results suggested that the EDC-NHS linker had conjugated with PLGA and Cy5.5.

PCS nanoparticle cell labeling and viability

After the cells were labeled with the nanoparticles at different concentrations, the dose dependent viability of the cells was assessed. Cells labeled with different concentrations showed no significant change in viability; cells labeled with 40 µg/mL of nanoparticles had more than 90% viability; thus, it was chosen as the labeling concentration (Fig. 2a). PCS nanoparticles internalized in MSCs were observed by confocal microscopy (Zeiss, LSM800). Green fluorescence indicates MSCs tagged with GFP; nuclei, stained with 4′,6-diamino-2-phenylindole (DAPI), can be observed in blue; and red fluorescence shows a PCS nanoparticle tagged with cyanine5.5 (Fig. 2b). Preservation of MSC stemness, after internalization of the nanoparticles, was also evaluated. PCS nanoparticle labeled cells were differentiated into multiple cell types using mouse mesenchymal cell functional identification kit (bio-techne®, R&D Systems, MN, USA). As shown in Fig. 2c, PCS nanoparticle labeled MSCs were able to differentiate into adipocytes, and osteocytes, indicating that the nanoparticles did not change the ability of the stem cells to differentiate into multiple mesenchymal lineages (Fig. 2c).

Fig. 1.

Characterization of PCS nanoparticles. a Schematic illustration of PCS nanoparticle preparation. b Hydrodynamic diameter of PCS. c Zeta-potential of PCS. d SEM image of PCS. e TEM image of PCS. f Fe ion measurement in PCS nanoparticle solution. g FT-IR analysis of PLGA and PLGA-Cy5.5

Immunofluorescence imaging of in vitro magnetic attraction

The Fe in the PCS nanoparticles core possesses the property of magnetization. MSCs labeled with the PCS nanoparticles were attracted with a magnet to determine the enhancement of migration capacity. MSCs were cultured in a 1-well culture dish, labeled, and a magnet was attached to one side. After 24 h incubation and 48 h incubation, the cells were fixed and DAPI stained and fluorescence images were captured at 1 mm intervals from the magnet (Fig. 3a). A total of 50% of cells adhered to 0–5 mm distance from the magnet, 30% adhered to 5–10 mm distance from the magnet, and 10% adhered to 10–15 mm distance from the magnet (Fig. 3b). Thus, an increased number of cells was observed in the area near the magnet; the migration of the PCS nanoparticle labeled cells could be manipulated by placing an external magnet.

Fig. 4.

Transwell migration assay of MSC; chemoattraction and magnetic attraction; a, d Control, b e MSC labeled with PCS nanoparticle and SDF-1 in lower chamber c, f MSC labeled with PCS nanoparticle and SDF-1 and magnet exposure for 24 h

Migration assay

Transwell migration assays were performed to further confirm the migratory capacity of the PCS nanoparticle labeled MSCs and to observe their chemotactic movement. As we have previously described, SDF-1/CXCR4 chemotaxis is an important homing factor, and this chemoattractant is responsible for the recruitment of stem cells to the injured site. Thus, chemotaxis and magnetic manipulation would increase the number of stem cells migrating to the desired site. We administered different concentrations of SDF-1 to induce chemotaxis; Fig. 4a–c shows that with increasing concentrations of SDF-1, the number of cells migrating across the membrane increased threefolds. The MSCs were labeled with PCS nanoparticles and the chemoattractant was provided in the lower well; the group with SDF-1 had the highest number of migrated cells across the membrane (Fig. 4d, e). This result indicated that nanoparticles, in the presence of a magnet, could increase homing efficiency of stem cells.

Fig. 5.

ABR (Auditory brainstem response) of ototoxic mouse model and FOBI analysis. a ABR test compared pre-injection to post-injection of ototoxic drug. b biodistribution of MSC after administration. c Mean fluorescence intensity analysis among experimental groups

Ototoxic animal model and abr recording

Auditory brain stem response was recorded 1 day prior to the ototoxic drug administration, to record the normal hearing threshold of the test subjects; click stimulus was delivered to the animals and stimuli response was recorded at 10–20 dB. The auditory brainstem response recordings were repeated 7 days after the administration of the ototoxic drugs—kanamycin and furosemide. The auditory brainstem response of the subjects was recorded at a stimulus intensity of 60–80 dB (Fig. 5a). The results indicated that the hearing threshold of the subject increased, demonstrating there is an event of hearing loss on which the animal model was formed.

Fig. 6.

Biodistribution of MSC-GFP in vivo ototoxic mouse model; reverse transcriptase PCR results quantifying GFP levels in the different tissue samples

Fluorescence imaging of organs, ex vivo

The biological properties of PCSs labeled MSCs were evaluated in the C57/BL6 ototoxic mouse model. Mice were injected with MSCs; (1) intravenous (IV) injection with magnet, (2) intratympanic (IT) injection with magnet, and (3) IT injection without magnet. Major organs and tissues were harvested at 24 h after injection and GFP fluorescence was measured by FOBI. The fluorescence of major organs was normalized, as mentioned in the method section. In the IT injection with magnet group, the highest mean fluorescence intensity (MFI) of GFP was observed in the brain (45 ± 0.44%) after injection. The MFI detected in the brain was fourfolds higher than in the liver (Fig. 5b). In the IT injection without magnet group, the highest MFI of GFP was observed in the brain (45 ± 6.8%). However, in the IV injection with magnet group, the highest MFI of GFP was observed in the liver. Thus, the injection route is significant in the biodistribution of MSCs. In addition, there were no significant differences between the IT injection groups (Fig. 5c), suggesting that 24 h (after injection) was insufficient to validate the magnetic attraction of MSCs to the brain.

Quantitation of GFP levels in different tissue samples

Biodistribution of the MSC’s were further investigated using reverse transcriptase PCR (RT-PCR). Tissue samples from different organs (liver, kidney, heart, lung, brain, cochlea) were collected and have quantified the expression level of GFP (Fig. 6). Expression of GFP’s were observed in different tissue samples however there was a significant increase in the expression level in the cochlea and brain samples. This results suggests that areas near the application of the magnet would attract more number of PCS nanoparticle labeled MSC.

Fig. 7.

Fluorescent microscopic images of biodistribution of MSC-GFP In vivo ototoxic mouse model. A Average number of green fluorescence expressing cells observed in the samples according to study group. B Fluorescent images of GFP expressing MSC; a–c Liver, d–f kidney, g–i brain, j–l cochlea

^PCSMSC transplantation and fluorescence imaging

PCS nanoparticle labeled MSCs were injected through the tail vein and intra-tympanically; 24 h post administration the mouse was euthanized and tissue samples were processed. Fluorescence microscopy images of the liver, lungs, kidney, heart, cochlea, and brain were obtained. In the tail vein administered group, with magnet application, the highest number of cells was observed in the liver followed by the brain, kidney, and cochlea. In the intra-tympanically administered group, with magnet application, the highest number of cells was observed in the brain followed by the liver, cochlea, and kidney; in the without magnet application group, a similar distribution pattern was observed, although with a lesser number of cells, when compared to that in the group where the magnet was applied (Fig. 7). Thus, application of an external magnet could increase the migratory capacity of ^PCSMSC toward the magnetic field, however the duration of magnet application and route of stem cell delivery could give better outcome of the results.

Fig. 2.

MSCs internalization by PCS. A Cytotoxicity test of PCS nanoparticle in concentration and time dependent manner. B Mesenchymal stem cell labeled with 40 ug/mL YRB for 24 h. a DAPI, b GFP, c Cy-5.5 in PCS nanoparticle, d merge (scale bar: 20 μm). C Differentiation of MSCs into adipocyte and osteocyte. a control (without adipogenic factors) b adipogenic differentiation without PCS c adipogenic differentiation with PCS (blue: DAPI, green: mFABP4). d control (without osteogenic factors) e osteogenic differentiation without PCS f osteogenic differentiation with PCS (blue: DAPI, green: mOsteopontin) (scale bar: 50 μm)

Discussion

More than 5% of the world population lives with some degree of hearing impairment (360 million people, including 32 million children) [14]. The main factors behind hearing degeneration are ototoxic drugs, aging, continued exposure to excessive noise, and infections. Upon irreversible damage of auditory system, the external devices of sound amplification, such as hearing aids and cochlear implants, and pharmacological therapies have shown little success [15, 16]. Therefore, many studies have focused on developing stem cell-based treatments to restore hair cells and spiral ganglion populations [17]. Among them, stem cell migration to the damaged cells, known as homing, is an important part of stem cell-based research. Human MSCs communicate with other cells in the body and appear to ‘home’ to the injured tissue in response to cellular damage signals, known as homing factors. Stem cell homing and targeted delivery post transplantation must be understood to increase the success rate of stem cell therapy in the inner ear. The fate of nanoparticle labeled stem cells should be evaluated to find an effective delivery method. The SPION, as an MRI contrast agent, has become popular in stem cell studies because it allows tracking of the cells and has been approved for safety. Commercially available iron oxide nanoparticles have been applied in different studies [18–20]. Many commercially synthesized iron oxide nanoparticles are available: Feridex® or Endorem™ (Advanced Magnetic, Cambridge, MA, USA), Resovist® (Bayer Schering Pharma AG, Berlin, Germany), fluidMag (chemicell, Berlin, Germany), and Molday ION Rhodamine B (BioPAL, Inc., Waltham, MA, USA) [8]. In this study, we have fabricated poly clustered superparamagnetic nanoparticles, which consist of a PLGA outer shell and an iron oxide inner core. The prominent feature of the PCS nanoparticles is that the PLGA coating improved the physiological stability of the nanoparticle, with the surface ζ-potential was − 29.6 mV, which improved its compatibility with the MSC. The surface charge of the nanoparticle affects the viability of the cells as the surface of the cells is also negatively charged. In vitro experiments were performed to confirm the biocompatibility of the PCS nanoparticles and the MSCs; internalization of the nanoparticles in the MSCs did not affect cell viability (Fig. 2a). Concerning nanoparticle structural characteristics, the absolute amount of Fe used in each experiment is important for determining the magnetic force. We have observed that the cellular uptake of nanoparticles in concentrations more than 40 µg/mL did not enhance the labeling efficacy.

The selection of targets, and chemokines and their receptors, such as CXCR4 and SDF-1, are important factors that induce homing of MSCs [21, 22]; combined effect of chemotaxis and magnetic attraction would maximize the homing phenomenon of stem cells. As demonstrated by Ju et al. [7], MSCs labeled with nanoparticles and increased expression of the CXCR4—a chemokine receptor found on the surface of MSCs, showed increased homing. To determine enhancement of migration by chemotactic signaling, we performed transwell migration assay, by supplementing the cells with SDF-1. Differences in the number of cells that migrated the transwell membrane, in the presence and absence of SDF-1 and magnet, were observed. A 2.5-fold increase in the number of migrated cells was seen, compared with those in the control and the SDF-1 and magnet applied groups. These results show that the MSCs labeled with PCS nanoparticles activated CXCR4, which increased their sensitivity to SDF-1, and could also magnetically attract the MSCs towards the magnetic field. Metal oxides have been shown to induce oxidative stress and can demonstrate reactive oxygen species (ROS) generation via Fenton-type reactions in MSCs labeled with nanoparticles [23]. CXCR4 and SDF-1 are upregulated under high ROS concentrations and/or hypoxic environments [24]. Lin et al. also described that CXCR4 induced ROS production in hematopoietic stem cells [25]. Thus, we need to devise strategies for safer design and manufacture of nanoparticles for stabilizing nanoparticle-induced ROS in magnetic attraction of MSCs with SPION.

Ahn et al. [8] suggested that to increase the efficacy of stem cell homing, magnetic targeting could increase the guidance of nanoparticle labeled stem cells to the target site. Magnetic attraction of magnetized labeled MSCs has been applied in ischemic heart disease [26, 27] and ischemic brain disease [28] and spinal cord [29], muscular injury [30], and skin [20], ocular [18], and olfactory injury [2]. Cheng et al. [27] placed a 1.3 T NdFeB magnet above the heart, inducing myocardial infarction in rat. They reported an increase of approximately 6.4-fold recovery in the MSC group with the magnet than in the control group with no treatment. In this study, we have guided PCS nanoparticle labeled stem cells to the cochlea and brain of an ototoxic animal model. Systemic delivery of stem cells showed the highest number of cells in the cochlea and the brain of the ototoxic animal model. Thus, stem can be delivered to the target site with the application of nanoparticles and magnet. However, the concentration of cells used for transplantation, magnet exposure time, and location of magnet needs further investigation to increase the efficiency of stem cell homing. Our study demonstrated that laboratory synthesized PCS nanoparticles, together with MSCs, could synergistically enhance stem cell delivery. Further research needs to be performed to increase the efficiency of stem cell therapy.

Conclusions

The SPION labeled MSC has great potential for stem cell therapy. We have successfully synthesized nanoparticles in accordance with the factors that could affect the efficacy of labeling in the cells. The PCS nanoparticles did not adversely affect the cells and had high efficacy in cellular internalization. The SPION allows guidance of stem cells to the targeted area through magnetic attraction. In this study we have observed that there was an increased number of cells retained in the areas applied with magnetic field. These results also suggest that magnetic force exerted in the target area would increase the enhancement of the stem cell attraction. The concentration of stem cell administration to lead therapeutic effect, strength of magnetic field application, needs further study to provide and develop treatment option for the diseases occurring in areas with anatomic microstructure and limited vasculature.

Abbreviations

PCS nanoparticle

Poly clustered superparamagnetic iron oxide nanoparticles

PLGA

Poly lactic-co-glycolic acid

Cy5.5

Cyanine 5.5

MSC

Mesenchymal stem cell

SDF-1

Stromal cell derived factor 1

CXCR4

Chemokine receptor type 4

Author contributions

YJA and WSY: equally contributed in conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; JSC: data analysis and interpretation; WCK: assembly of data, data analysis and interpretation; SHL: collection and assembly of data, data analysis and interpretation; DJP: data analysis and interpretation; JEP: collection and assembly of data, data analysis and interpretation; JK: conception and design, administrative support, data analysis and interpretation, final approval of manuscript; YJS: administrative support, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.

Compliance with ethical standards

Conflict of interest

Author Ahn, Yeji declares that she has no conflict of interest. Author Yoon, Wan Su declares that he has no conflict of interest. Author Choi, Jin Sil declares that she has no conflict of interest. Author Kim, Woo Cheol declares that he has no conflict of interest. Author Lee, Su Hoon declares that he has no conflict of interest. Author Park, Dong Jun declares that he has no conflict of interest. Author Park, Jeong Eun declares that she has no conflict of interest. Author Key, Jaehong declares that he has no conflict of interest. Author Seo, Young Joon declares that he has no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Human and animal rights

This article does not contain any studies with human participants performed by any of the authors.