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Back to Journal »International Journal of Nanomedicine» Volume 16

Superparamagnetic iron oxide nanoparticles can guide the neurite extension and orientation of spiral ganglion neurons in a magnetic field

Authors Hu Y, Li D, Wei H, Zhou S, Chen W, Yan X, Cai J, Chen X, Chen B, Liao M, Chai R, Tang M

Published on July 2, 2021, the 2021 volume: 16 pages 4515-4526

DOI https://doi.org/10.2147/IJN.S313673

Single anonymous peer review

Editor approved for publication: Professor Israel (Rudi) Rubinstein

Yangnan Hu,1,* Dan Li,2,* Hao Wei,3,* Shan Zhou,1 Wei Chen,1 Xiaoqian Yan,1 Jaiying Cai,1 Xiaoyan Chen,1 Bo Chen,4 Menghui Liao,1 Renji Chai,1 ,5 Tang Mingliang1,5,6 1 State Key Laboratory of Bioelectronics, School of Life Science and Technology, School of Life Science and Technology, Southeast University, Jiangsu Province Key Laboratory of Biomedical Research, Nanjing, 210096; 2 Hefei University Biofood School of Science and Environment, Hefei 230601; 3 Department of Otorhinolaryngology, Head and Neck Surgery, Drum Tower Clinical School, Nanjing Medical University, Nanjing 210000; 4 Institute of Materials Science and Devices, University of Science and Technology of Suzhou, Suzhou 215009; 5 Collaborative Innovation Center for Neural Regeneration, Nantong University, Nantong 226001; 6 Institute of Cardiovascular Surgery and Cardiovascular Science, First Affiliated Hospital of Soochow University School of Medicine, Suzhou, 215000 *The above authors have equal contributions to this research Corresponding author: Chai Renjie; Mingliang Tang Email [email protected]; [Email protection] Introduction: Neurology Regeneration is a major challenge in the field of neuroscience to treat degenerative diseases and repair damaged nerves. Many studies have shown the importance of physical stimulation for neuron growth and development. Here we report a method of using superparamagnetic iron oxide (SPIO) nanoparticles and magnetic field (MF) to physically guide neuron orientation and neurite growth. Methods: The SPIO nanoparticles were synthesized by the classical chemical co-precipitation method, and then characterized by transmission electron microscopy, dynamic light scattering, and vibrating sample magnetometer. CCK-8 assay and LIVE/DEAD assay were used to determine the cytotoxicity of the prepared SPIO nanoparticles and MF. The immunofluorescence image was captured by a laser scanning confocal microscope. A wound healing test was used to assess cell migration. Results: The prepared SPIO nanoparticles have narrow size distribution, low cytotoxicity, and superparamagnetic properties. SPIO nanoparticles coated with poly-L-lysine can be internalized by spiral ganglion neurons (SGN) and have no cytotoxicity at concentrations below 300 μg/mL. After internalizing SPIO nanoparticles with or without external MF, the neurite extension of SGNs was promoted, which may be due to the promotion of growth cone development. It was also confirmed that SPIO can regulate cell migration and can preferentially guide neurite outgrowth in SGN along the direction of external MF application. Conclusion: Our results provide a basic understanding of the regulation of cell behavior under physical cues and propose an alternative treatment for sensorineural hearing loss caused by SGN degeneration. Keywords: physical cues, neurite orientation, hearing loss, cochlear implant, migration

In the mammalian auditory system, the spiral ganglion neuron (SGN) is the main neuron that transmits sound information from the mechanosensory hair cells located in the inner ear to the cochlear nucleus in the brainstem. 1 Degeneration of SGN due to loss of hair cells is the main sensorineural hearing loss, but SGN can also degenerate due to overexposure to acoustics, 2,3 ototoxic drugs, 4,5 and aging. Ears, leading to permanent sensorineural hearing loss. Therefore, SGN is a potential target for protection and regenerative therapy. 8 In addition to pharmacological methods for the treatment of hearing loss, cochlear implant (CI) is an effective clinical option for partial restoration of hearing in patients with hearing loss. However, the functional principle of CI is the direct electrical stimulation of SGN, which means that the effectiveness of CI mainly depends on the remaining SGN. 6,7 Another factor that limits the effectiveness of CI is the anatomical gaps and SGNs that exist between the electrode arrays, leading to current diffusion and non-specific stimulation. 9-12 One of the potential solutions to this is to direct the neurites of SGNs to grow toward the stimulation electrodes. 13 Therefore, it is important to maintain and promote survival and growth of SGN neurites and to guide them toward the goal to improve the CI performance of patients with sensorineural hearing loss.

There is increasing evidence that physical stimulation is a key factor in the growth and development of neurons,14,15 and single cells like neurons can sense and respond to the mechanics of their environment. 16,17 In recent years, magnetic nanoparticles have been widely used in the field of biomedicine for magnetic resonance imaging (MRI), 18-20 cell labeling, 21,22 and targeted drugs or gene delivery. 23 Magnetic iron oxides, such as Fe3O4 and c-Fe2O3 nanoparticles are the most studied particles. These applications are due to their high magnetization under a magnetic field, high stability of 24-26, and biocompatibility. 27,28 More importantly, recent studies have demonstrated that superparamagnetic iron oxide (SPIO) nanoparticles have the ability to direct neurites. 29, 30

In this study, we tried to demonstrate the potential of SPIO nanoparticles and magnetic fields (MF) to regulate SGN, especially in terms of neuron orientation and axon growth.

The SPIO nanoparticles used in this study were synthesized by the classical chemical co-precipitation method as described earlier. 31 In short, an aqueous solution (10 ml) of polydextrose sorbitol carboxymethyl ether (PSC, 200 mg) was blown into nitrogen for 5 minutes to remove oxygen. Next, 30 mg FeCl2 (0.236 mmol) and 60 mg FeCl3 (0.37 mmol) were completely dissolved in 15 mL of deionized water, and the mixture was added to the PSC solution. Subsequently, 1 g of ammonium hydroxide (28% w/v) was quickly added to the mixed solution, and vigorously mechanically stirred in a water bath at 80°C for 30 minutes. Finally, an ultrafiltration centrifuge tube was used to collect the iron oxide nanoparticles and washed with ultrapure water several times. Transmission electron microscope (TEM; JEM-2100, JEOL, Japan) and dynamic light scattering were used to determine the morphology and size of the prepared SPIO nanoparticles. The magnetic properties of SPIO nanoparticles were measured at 37°C using a vibrating sample magnetometer. The XRD pattern was obtained from an X-ray diffractometer (Rigaku Ultima IV, Japan). In order to absorb SPIO nanoparticle cells into SGN, the prepared nanoparticles were coated with poly-L-lysine (Sigma, USA) in an ultrasonic bath.

All animal protocols were approved by the Institutional Animal Care and Use Committee of Southeast University, and all animals were treated in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines. All FVB mice are bred internally under a 12:12 hour light-dark cycle. All efforts are made to minimize the number of animals used and reduce their suffering.

To obtain SGN, the mice on the 2nd or 3rd day after birth were decapitated, and the brains were removed and immersed in ice-cold sterile Hanks' Balanced Salt Solution (HBSS; Gibco, USA) and then the temporal bones were dissected. Isolate the cochlea from the temporal bone in the HBSS under a dissecting microscope (Leica, Germany), cut off the bone shell, and remove the stria vascularis and the organ of Corti. The snails were collected and placed in 0.1% trypsin (Gibco, USA) and digested at 37°C for 10 minutes. Add soybean trypsin inhibitor (Gibco, USA) to stop digestion. Isolate the cells by gently pipetting up and down 80-100 times with a blunt tip, and filter the solution through a 40 µm cell strainer (Falcon, USA) to obtain a single cell suspension. Before cell seeding, the coverslips were coated with laminin (Sigma, USA) in phosphate buffered saline (PBS; Gibco, USA) at 37°C overnight, and then washed with PBS. SGN was spread on a laminin-coated coverslip and incubated in a 5% CO2 humidified incubator at 37°C for future experiments. During the first 24 hours, SGN was cultured in SGN1 medium, which consisted of DMEM/F12 (Gibco, USA), 5% fetal bovine serum (Invitrogen, USA) and ampicillin (50 µg/mL; Sigma, USA) composition. For long-term culture, replace the SGN1 medium with DMEM/F12, N2 (Invitrogen, United States), B27 (Invitrogen, United States), EGF (20 ng/mL, Peprotech, United States), FGF (20 ng/mL, Peprotech, United States), IGF (50 ng/mL, Peprotech, United States), HSP (20 ng/mL, Sigma, United States), and ampicillin (50 µg/mL, Sigma, United States).

MF is produced by two rectangular neodymium magnets. The magnets are placed parallel to both sides of the box, and a 35 mm plate is placed in the middle of the box (Figure S1, supporting information). We believe that the most central area of ​​a petri dish with a diameter of 10 mm receives roughly the same MF intensity and direction. Adjust the MF intensity in the central area by using magnets of different sizes. The strength of the MF formed by the magnet is measured with a Teslameter (HT20).

Cell counting CCK-8 kit (Beyotime, China) and LIVE/DEAD viability/cytotoxicity kit (Invitrogen, USA) were used to determine the cytotoxicity of the prepared SPIO nanoparticles and MF. In the CCK-8 test, SGN was seeded in a 96-well plate coated with laminin at a concentration of 1×104 cells/well. After adhesion, the cells were exposed to SPIO nanoparticles (concentrations of 0, 50, 100, 200, 300, and 500 μg/mL) for 6, 24, and 36 hours. After washing with medium for 3 times, add 10 μL of CCK-8/200 μL of fresh medium to each well, avoid light at 37°C for 30 min, and measure the absorbance at 450 nm with a Bio-Rad microplate reader to assess relative cell viability. In the LIVE/DEAD test, SGN is seeded on laminin-coated coverslips for adhesion. The cells are then exposed to 100 μg/mL SPIO nanoparticles, MF (80–90 mT), or both for 3 days. According to the manufacturer's instructions, use the LIVE/DEAD Viability/Cytotoxicity Kit to assess cell viability. The working concentrations of calcein AM and ethidium homodimer-1 are 2 μM and 0.5 μM, respectively.

To confirm cellular iron uptake, SGN was incubated with SPIO nanoparticles for 24 hours for Prussian blue staining. After incubation, the medium containing SPIO nanoparticles was removed, and SGN was washed 3 times with PBS to remove as many SPIO nanoparticles as possible from the cell surface. After being fixed in 4% paraformaldehyde for 45 minutes at room temperature, the cells were incubated in a Prussian blue staining solution (1:1 mixture of hydrochloric acid and potassium ferrocyanide) (Sigma, USA) for 30 minutes to pass an optical microscope Assess the intracellular iron distribution (Leica). After incubation with SPIO nanoparticles labeled with Rhodamine B (RB-SPIO), fluorescence microscopy images of the cells were also used to evaluate the absorption of SPIO nanoparticles. In short, after the above incubation, washing and fixation, the cells were stained with DAPI for 30 minutes at room temperature, and finally washed with PBS 3 times. Confocal imaging was performed on a Zeiss 700 laser scanning confocal microscope (LSM700). The quantification of SPIO uptake by SGN is determined by the iron content of the particles in the cells. The iron content is measured by inductively coupled plasma mass spectrometer (ICP-MS, Thermo).

SGN was seeded in a petri dish pre-coated with laminin, and the cells were treated with 100 μg/mL SPIO nanoparticles 24 hours after seeding. When treating cells with MF, place the petri dish in the box between the two magnets, as described above. After 7 days, the length of the axons and the angle between the direction of each axon and the MF were measured. The neurite orientation is quantified as an orientation index, which is defined as Oi = cos(θ) (0<θ<π/2). Use Image Pro Plus software for analysis.

After incubation, the SGN was washed once with PBS and fixed with 4% paraformaldehyde in PBS at room temperature for 45 minutes, and then washed 3 times with PBS supplemented with 0.1% Triton-X100 (Solarbio, China) (PBST). The cells were then blocked in 1% bovine serum albumin (BSA; Solarbio, China) dissolved in PBS/0.1% Triton-X100/10% heat-inactivated donkey serum (Solarbio, China) at room temperature for 1 hour, and then Stay overnight at 4°C in blocking buffer with primary antibodies, including mouse anti-βIII-tubulin (Abcam, USA), rabbit anti-EEA1 (Abcam, USA), rabbit anti-Rab7 (CST, USA) and mouse anti-LAMP2 (CST, United States). On the next day, SGNs were washed 3 times with PBST. Appropriate Alexa Fluor conjugated secondary antibodies and DAPI or phalloidin are incubated in PBS/0.1% Triton X-100/1% BSA at room temperature for 1-2 hours, and then washed with PBST 3 times. Finally, cover the cells with a cover glass in DAKO fluorescent mounting medium and observe under a Zeiss 700 laser scanning confocal microscope (LSM700).

A wound healing test was used to assess cell migration. 32 In short, SGN was seeded in a 60 mm Petri dish and grown to form a cell monolayer. Then use a sterile 200 μL pipette tip to create a scratch wound. Wash off floating cells with PBS, and then culture in fresh serum-free medium. Images were taken under an optical microscope at 0, 12, and 24 hours after the scratch. The percentage of wound closure was calculated from three independent experiments.

Use ImageJ or Image Pro Plus software to analyze the confocal and brightfield images of SGN to evaluate the cell length, direction, migration, growth cone and synaptic growth. All statistical analyses are performed in GraphPad Prism, and all data are shown as mean ± SD. Use one-way analysis of variance for statistical analysis, followed by Dunnett's multiple comparisons (used to compare more than two groups) or pass the two-tailed unpaired Student's t-test (used to compare two groups). A value of p <0.05 is considered statistically significant. All experiments are carried out at least 3 times.

SPIO nanoparticles are synthesized by coating γ-Fe2O3 core with PSC. The size and morphology of SPIO nanoparticles are determined by TEM and dynamic light scattering. The SPIO nanoparticles are well dispersed without agglomeration, and the size distribution of the core is about 6-8 nm (Figure 1A and B). The hydrodynamic size of SPIO nanoparticles is approximately 21 nm (Figure 1C). The XRD spectrum (Figure S2, supporting information) shows the characteristic peaks, which are located at 30.3, 35.7, 43.6, 53.4, 57.2 and 63.1, which are assigned to (220), (311), (400), (422), (511) )) And (440) phases of γ-Fe2O3 crystals (JCPDS: 39–1346), consistent with another study. 33 The magnetic properties of SPIO nanoparticles are essential for studying their joint regulation of cells after applying MF. The results of the vibrating sample magnetometer show that the SPIO nanoparticles obtained are superparamagnetic and the saturation magnetization is about 56 emu/g (Figure 1D). Figure 1 Characterization of the prepared SPIO nanoparticles. (A) TEM image of SPIO nanoparticles. (B) Size distribution of SPIO nanoparticles in TEM image. (C) The hydrodynamic size of SPIO nanoparticles. (D) Representative magnetization curve of SPIO nanoparticles.

Figure 1 Characterization of the prepared SPIO nanoparticles. (A) TEM image of SPIO nanoparticles. (B) Size distribution of SPIO nanoparticles in TEM image. (C) The hydrodynamic size of SPIO nanoparticles. (D) Representative magnetization curve of SPIO nanoparticles.

We next explored the absorption of SPIO nanoparticles by SGNs. Prussian blue staining and fluorescence confocal images verified the internalization of SPIO nanoparticles into SGNs (Figure 2A and B). Nanoparticles accumulate in the cell body and neurites, but not in the nucleus. Next, we quantitatively evaluated the internalization of SPIO in terms of incubation time and dose by ICP-MS. We found that the amount of iron increased with increasing incubation time and SPIO concentration (Figure 2C), indicating that SGN internalized SPIO nanoparticles in a time- and dose-dependent manner. Figure 2 Internalization of SPIO nanoparticles by SGN. (A) Prussian blue staining of SGN incubated with (right) and without (left) 100 µg/mL SPIO nanoparticles for 24 hours. (B) Representative confocal images of SGN incubated with (bottom) and without (top) 100 μg/mL RB-SPIO nanoparticles for 24 hours. (C) A graph of the cellular uptake and incubation time and concentration of SPIO nanoparticles, determined by ICP.

Figure 2 Internalization of SPIO nanoparticles by SGN. (A) Prussian blue staining of SGN incubated with (right) and without (left) 100 µg/mL SPIO nanoparticles for 24 hours. (B) Representative confocal images of SGN incubated with (bottom) and without (top) 100 μg/mL RB-SPIO nanoparticles for 24 hours. (C) A graph of the cellular uptake and incubation time and concentration of SPIO nanoparticles, determined by ICP.

In order to verify the biocompatibility of SPIO nanoparticles, SGN was used for CCK-8 determination. These SGNs were incubated with SPIO nanoparticles at a concentration of 0–500 μg/mL for 6, 12, 24, and 36 hours. The concentration of SPIO nanoparticles below 300 μg/mL showed low cytotoxicity (> 80% cell viability) (Figure 3A). However, when incubated at a high concentration of 500 μg/mL, SPIO nanoparticles showed cytotoxic effects in SGN. At a concentration of 100 μg/mL, the uptake of SPIO nanoparticles was sufficient and did not have any significant effect on SGN activity even after 36 hours of further incubation, so we used a concentration of 100 μg/mL in all experiments. Since the subsequent experiment introduced a magnetic field, we next tested the cytotoxicity of SPIO nanoparticles and a magnetic field (SPIO MF) to SGN. CCK-8 and LIVE/DEAD staining showed that SPIO, MF and SPIO MF did not induce significant cell death in SGN (Figure 3B and C), indicating that SPIO, MF and SPIO MF are biocompatible and non-toxic. Figure 3 Cell viability of SGN treated with SPIO nanoparticles and MF. (A) The cell viability results measured by CCK-8 after incubating SGN with 100 μg/mL SPIO nanoparticles at different concentrations for different times. * p <0.05 passed the student's t test. (B) The cell viability of control SGNs and SGNs treated with SPIO nanoparticles (100 μg/mL), MF (80–90 mT) or SPIO MF was evaluated by LIVE/DEAD cell assay. Live cells are stained green, and dead cells are stained red. (C) Percentage of live cells in different groups in LIVE/DEAD test. One-way analysis of variance showed that there were no significant differences between the groups.

Figure 3 Cell viability of SGN treated with SPIO nanoparticles and MF. (A) The cell viability results measured by CCK-8 after incubating SGN with 100 μg/mL SPIO nanoparticles at different concentrations for different times. * p <0.05 passed the student's t test. (B) The cell viability of control SGNs and SGNs treated with SPIO nanoparticles (100 μg/mL), MF (80–90 mT) or SPIO MF was evaluated by LIVE/DEAD cell assay. Live cells are stained green, and dead cells are stained red. (C) Percentage of live cells in different groups in LIVE/DEAD test. One-way analysis of variance showed that there were no significant differences between the groups.

In order to verify our hypothesis that SPIO nanoparticles can stimulate neuron growth and neurite orientation under external MF, SGN is treated with SPIO nanoparticles, MF (20-30 mT, 50-60 mT, 80-90 mT) or SPIO MF . SGNs were harvested after 7 days of culture (DIV7) and immunostained with anti-βIII tubulin (Figure 4A). The neurite length and the angle between the neurite and the MF direction were measured to compare the neurite extension and orientation ability of SGN in different groups. After 7 days of culture, regardless of the MF intensity, the average neurite length of SPIO and SPIO MF groups was longer than that of the control group (Figure 4B and Figures S3A and S3B, S4A and S4B, supporting information). In order to quantify the direction of the axon, the angle between the long axis of the axon and the MF direction was measured, and the direction index was defined as Oi = cosθ (0 <θ <π/2). When the MF intensity is 50-60 mT and 80-90 mT, the angular distribution (0°) between axons and MF indicates that the SGN axons in the SPIO MF group are preferentially aligned along the MF direction, while in other groups, the nerve Highlight the random distribution in the direction of no preference (Figure 4C and Figure S4C, supporting information). However, when the MF intensity was 20-30 mT, even in the SPIO MF group, the angular distribution between neurites and MF did not show the preferred direction (Figure S3C, supporting information). Figure 4 SPIO nanoparticles (100 μg/mL) and MF (80–90 mT) promote the extension and orientation of SGN neurites. (A) Representative fluorescence images of different processed SGNs. (B) Average neurite length. ***p <0.001, ****p <0.0001 through one-way analysis of variance. (C) Axon orientation index (cosθ). For neurites along the magnetic direction, Cos = ~1. * p <0.05 One-way analysis of variance.

Figure 4 SPIO nanoparticles (100 μg/mL) and MF (80–90 mT) promote the extension and orientation of SGN neurites. (A) Representative fluorescence images of different processed SGNs. (B) Average neurite length. ***p <0.001, ****p <0.0001 through one-way analysis of variance. (C) Axon orientation index (cosθ). For neurites along the magnetic direction, Cos = ~1. * p <0.05 One-way analysis of variance.

We further determined the effects of SPIO nanoparticles and MF on SGN growth cone and synapse density by immunofluorescence. We stained the cultured SGN with the neuronal marker anti-β-tubulin, and stained the actin-supported growth cones with phalloidin in DIV3. Compared with the control, when treated with SPIO or SPIO MF, the growth cone area of ​​neurites is larger and the average filopodia length is longer (Figure 5A, B, and D). When only exposed to MF, there were no significant differences in growth cone area, number of filopodia, and average filopodia length compared with the control (p> 0.05, Figure 5B-D). There was no significant difference in the number of filopodia in any group (Figure 5C). Overall, our results indicate that SPIO and SPIO MF can significantly promote growth cone development, which may help enhance neurite extension. Figure 5 SPIO nanoparticles (100 μg/mL) and MF (80–90 mT) accelerate the development of SGN growth cones and filopodia. (A) Low-resolution and high-resolution confocal images of growth cones immunostained with βIII-tubulin (red) and phalloidin (green) to label the F-actin structure. (B) Average growth cone area. ***p <0.001, ****p <0.0001 through one-way analysis of variance. (C) The average number of filopodia emerging from growth cones. There is no significant difference in one-way analysis of variance. (D) The average length from the tip of each filopodium to the edge of the growth cone. **p <0.01, ****p <0.0001 through one-way analysis of variance.

Figure 5 SPIO nanoparticles (100 μg/mL) and MF (80–90 mT) accelerate the development of SGN growth cones and filopodia. (A) Low-resolution and high-resolution confocal images of growth cones immunostained with βIII-tubulin (red) and phalloidin (green) to label the F-actin structure. (B) Average growth cone area. ***p <0.001, ****p <0.0001 through one-way analysis of variance. (C) The average number of filopodia emerging from growth cones. There is no significant difference in one-way analysis of variance. (D) The average length from the tip of each filopodium to the edge of the growth cone. **p <0.01, ****p <0.0001 through one-way analysis of variance.

Synapses mediate the transmission of information between neurons, and their structure and function directly determine neurobehavior. Therefore, the influence of SPIO nanoparticles and MF on synaptic density was examined in DIV14. Synapsin-1 and PSD95 have been shown to play an important role in synaptic plasticity and synaptic maturation, 34 and the presynaptic and postsynaptic regions are treated with antibodies against the presynaptic protein synapsin 1 and the postsynaptic protein PSD95. Immunostaining. Both low-magnification and high-magnification laser confocal microscopy showed extensive expression of synapsin and the co-localization of synapsin-1 and PSD95 in all groups (Figure 6A and B), which indicated that normal synaptic structures were generated. In addition, there was no significant difference in the synaptic density of SGN in the different groups (Figure 6C). Figure 6 SPIO nanoparticles (100 μg/mL) and MF (80–90 mT) have no effect on the synaptic density of SGN. Low resolution (A) and high resolution (B) representative confocal images of synapses immunostained for synapsin 1 and PSD95. (C) The average synapse density of SGN. There is no significant difference in one-way analysis of variance.

Figure 6 SPIO nanoparticles (100 μg/mL) and MF (80–90 mT) have no effect on the synaptic density of SGN. Low resolution (A) and high resolution (B) representative confocal images of synapses immunostained for synapsin 1 and PSD95. (C) The average synapse density of SGN. There is no significant difference in one-way analysis of variance.

Finally, we evaluated the effects of SPIO nanoparticles and MF on the migration of SGN. The wound healing test showed that compared with the control group, SPIO nanoparticles greatly inhibited wound closure. However, when MF was applied, the cell migration ability inhibited by SPIO internalization was greatly restored (Figure 7A and B). When only MF acts on SGN, their migration capabilities remain unchanged. Figure 7 SPIO nanoparticles (100 μg/mL) and MF (80–90 mT) regulate the migration behavior of SGN. (A) Wound healing test shows the effect of SPIO nanoparticles and MF on the migration of SGN. (B) Histogram of the percentage of wound closure in different groups. *p <0.05, ***p <0.001 by one-way analysis of variance.

Figure 7 SPIO nanoparticles (100 μg/mL) and MF (80–90 mT) regulate the migration behavior of SGN. (A) Wound healing test shows the effect of SPIO nanoparticles and MF on the migration of SGN. (B) Histogram of the percentage of wound closure in different groups. *p <0.05, ***p <0.001 by one-way analysis of variance.

Our results demonstrate the ability of SPIO nanoparticles and MF to support normal SGN survival and growth, promote neurite extension, enhance neurite alignment, and regulate cell migration in vitro. However, the biological safety of SPIO nanoparticles should be carefully evaluated before further clinical application. It is worth mentioning that we have completely followed the technology used to produce Ferumoxytol during preparation and synthesis-which is approved by the FDA for clinical applications The only inorganic nanomedicine. SPIO nanoparticles. More importantly, our synthesized SPIO nanoparticles have also been approved by the CFDA as MRI reagents and iron supplements, indicating that the SPIO nanoparticles we used in our research have very high biological safety. In this study, we proved that SPIO nanoparticles have good biocompatibility with SGN. In SGN cultured with SPIO nanoparticles, the nanoparticles are located in the cell body and neurites, and there is no cytotoxicity at low concentrations. Similar results have been reported in studies evaluating the potential cytotoxicity of SPIO nanoparticles in different types of cells. 31,35,36 Liu et al.’s work showed that when cells use 50 μg/mL SPIO nanoparticles,35 and our results show that SPIO nanoparticles are not cytotoxic to SGN at a concentration of 300 μg/mL, showing more Good biocompatibility. In fact, the cytotoxicity of SPIO nanoparticles varies with particle size and cell type, and is related to the type and stability of the nanoparticle coating. 37 The SPIO nanoparticles we used in this work were coated with poly-L-lysine before incubation with SGN, which should improve the biocompatibility of the nanoparticles.

Our results show that SPIO nanoparticles and MF promote the growth and alignment of neurites. SPIO nanoparticles have the ability to promote the extension of SGN neurites, and external MF is not a necessary condition for this. Although the underlying mechanism responsible for the promotion of neurite growth in nerve cells through SPIO nanoparticles is still unclear, it is speculated that Fe ions can be released from iron oxide nanoparticles in cells, thereby having the ability to promote neurite growth. 38 In addition, the combination of SPIO nanoparticles and external MF can guide neurites to preferentially grow parallel to the MF direction. Similar results were obtained in previous reports. Yuan et al. found that SPIO-Au core-shell nanoparticles functionalized with nerve growth factor can accelerate the axon growth of PC-12 cells and align the axon with the applied magnetic force. 39 The alignment of the axons can be attributed to the SPIO acting on the neurites by the magnetic field, which makes it possible to manipulate and orient the neurites to the desired orientation. In fact, more and more evidence shows that cell tension is a key factor in neuron growth and development. 40 The mechanical interaction between physical cues and cells seems to be not only the cause of neurite outgrowth, but also the cause of neurite direction. Our results also confirmed that MFs promote neurite outgrowth and guide the neurite outgrowth of SGN cultured with SPIO. However, due to the limitation of experimental settings, the MF intensity used in this study is not high enough. In the future, stronger MFs should be used to study whether they have a better targeting effect on cells. It is also useful to determine the minimum MF intensity that can produce the results observed in this study. We believe that using MF to grow SGN neurites towards CI electrodes will help promote direct contact between cells and implant electrodes. This improved connection will increase the effectiveness of CI as a treatment for sensorineural hearing loss.

The ability to control neuronal migration has important potential in nerve regeneration research and treatment. Therefore, we conducted a wound healing test to evaluate whether SPIO nanoparticles and MF affect the migration of SGN. We found that SPIO nanoparticles significantly inhibited the migration of SGN compared to untreated cells. However, compared with the SPIO group, the SGN with internalized SPIO nanoparticles showed enhanced migration when external MF was applied, which was comparable to the control group. Therefore, the reduced migration ability caused by SPIO nanoparticles can be reversed by applying external MF. In fact, the influence of SPIO nanoparticles on the migration of different types of cells is still controversial. Neural stem cells labeled with SPIO nanoparticles have been shown to reduce the cell’s ability to migrate, 41 which is similar to that observed in this study. According to the results of Soenen et al., when SPIO nanoparticles are internalized, the cellular actin cytoskeleton and microtubule network will be destroyed, resulting in loss of adhesion and reduced migration ability. 42 In contrast, another study showed that the internalization of SPIO did not have a significant effect on Schwann cell migration compared to the control, although the application of MF significantly enhanced Schwann cell migration to a magnetic source. 43

The SPIO nanoparticles used in this study were synthesized using a classic chemical co-precipitation method. These nanoparticles have been approved by the CFDA as MRI reagents and iron supplements, thus showing their high safety. The vitality measurement performed in this study confirmed the excellent biocompatibility of SPIO nanoparticles. We further proved that the mechanical tension generated by SPIO nanoparticles in MF can promote neuron growth and direct the direction of SGN neurites. We believe that future in vivo studies will reveal how nerve cells respond to complex mechanical environments. Combined with the previously published results, we believe that the results of this study are of great significance for nerve regeneration and recovery, thereby bringing new strategies for the treatment of nerve damage and degenerative diseases, especially for the treatment of sensorineural hearing loss. Due to the degradation of SGN.

The data can be obtained after reasonable request to the corresponding author (Dr. Tang Mingliang).

This work was funded by the National Major Basic Research and Development Program (973 Program) (2017YFA0104303) and the National Natural Science Foundation of China (81970883).

The authors report no conflicts of interest in this work.

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