Materials and animal care
Bovine serum albumin (BSA), fetal bovine serum (FBS), Dulbecco's modified Eagle medium (DMEM), neurobasal medium, NGF, penicillin/streptomycin, trypsin–EDTA, CellLight Golgi-RFP, MitoTracker Red, ER-Tracker Red, LysoTracker Red, Cytochalasin D (Cyto D), tubulin beta rabbit polyclonal antibody, goat anti-rabbit IgG secondary antibody, and antifade mountant with DAPI and Hoechst nucleic acid stains were obtained from Life Technologies Corporation (29851 Willow Creek Road, Eugene OR 97402, USA). Methyl-β-cyclodextrin (MβCD), genistein, chlorpromazine, amiloride, 2-deoxy-d-glucose (2-DG), and Cell Counting Kit-8 (CCK8) were purchased from Sigma-Aldrich (3050 Spruce Street, Saint Louis, MO 63103 USA). Rabbit anti-Cdh11, rabbit anti-Csf1r, mouse monoclonal anti-β-actin and anti-mouse antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, USA). The rabbit anti-Ppp1r1c antibody was obtained from Affinity (USA). The 35 mm imaging ibidi petri dishes (ibidi, 80156) were obtained from ibidi GmbH (Am Klopferspitz 19, 82152 Martinsried, Germany), and the two-compartment microfluidic chambers were purchased from Xona Microfluidics (Temecula, CA 92590, USA).
All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Jilin University and approved by the Animal Ethics Committee of Jilin University. All efforts were made to minimize the number of animals used and their suffering.
Synthesis and characterization of FMSPs
FMSPs were prepared as described in our previous work [13]. Briefly, oleic acid (OA)-capped Fe3O4 nanoparticles (NPs) were synthesized following the thermal decomposition method [14]. The average diameter of the as-prepared Fe3O4 NPs was 5.8 nm. Subsequently, OA-capped Fe3O4 NPs dispersed in toluene and water containing sodium dodecyl sulfate (SDS) and Rhodamine 6G were mixed to form an oil-in-water (O/W) microemulsion. Fe3O4 superparticles (SPs) with an average diameter of 50 nm were produced after the evaporation of toluene [15,16]. Finally, polydopamine (PDA) was coated on the surface of Fe3O4·Rhodamine 6G SPs via the oxidation polymerization of dopamine monomers under alkaline conditions [17].
The average size, distribution, and morphology of FMSPs were studied by high-resolution transmission electron microscopy (HRTEM) using a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV with a CCD camera. The hydrodynamic size and zeta potential of the FMSPs were measured through dynamic light scattering (DLS) by a Zetasizer Nano-ZS (Malvern Instruments). Magnetic property characterization of the samples was performed at 300 K in a superconducting quantum interference device (SQUID) magnetometer (MPMS-XL, Quantum Design, Inc., San Diego, CA). Fluorescence spectroscopy was performed with a Shimadzu RF-5301 PC spectrophotometer.
Cell and cell culture
To study the influence of magnetic manipulation on cellular behavior in neural cells, two neural cell types, rat adrenal pheochromocytoma cells (PC12, derived from a neuroendocrine tumor of the sympathetic nervous system, which is often used as a neuronal cell model) and primary rat dorsal root ganglia (DRG) neurons, were cultured. The PC12 cells used in this work were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10% FBS and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) [18,19]. Primary DRG neurons were isolated from Sprague-Dawley rats (age 1-3 days) as described previously [20,21] and maintained in neurobasal medium supplemented with 2% B27, 2 mM L-glutamine, 0.5% antibiotics (penicillin/streptomycin) and 50 ng/ml NGF. All cultures were conducted in an incubator at 37 °C in a humidified atmosphere with 5% CO2. The details of primary DRG neuron extraction are presented in Additional file 1: Supplementary Information.
PC12 cells were used for cellular behavior studies of the internalization of FMSPs, morphology analysis and examination of the magnetically guided outgrowth of neurites in directional orientation. DRG neurons were used as a model for examining magnetic tension achieved via FMSP interactions that stimulates the growth and elongation of axons at the single-cell level.
Cytotoxicity assay
The CCK8 assay was used to examine the cytotoxicity of FMSPs. PC12 cells were seeded in 96-well plates at 1×104 cells per well and incubated for 24 h. Different concentrations of FMSPs (0, 20, 50, 80, 100, 200 μg/ml) were added and incubated for another 24 h. Afterwards, the medium in each well was replaced with 90 μl of serum-free medium and 10 μl of CCK8. After 4 h of incubation, the absorbance of each well was measured at 450 nm using a Synergy HT microplate reader (Bio-Tek, Winooski, VT, USA). The survival rate of cells without FMSPs in the control wells was assumed to be 100%. Cell viability was derived through the absorbance percentage relative to the control cells.
Quantification of internal FMSPs per cell
The cells were seeded in 6-well plates (5 × 105 cells per well) and incubated for 24 h. After that, the cells were treated with FMSPs (10 μg/ml) for 4 h and washed twice with cold PBS containing 1 mM deferoxamine to remove iron that was nonspecifically attached to the cell membranes rather than taken up into the cells. Then, the cells were collected and counted. The amount of iron inside the cells in the samples was measured by ICP-AES measurements with a PerkinElmer Optima 3300DV. Based on the number of cells in the samples, we can calculate the average mass of iron per cell. From the density (ρ, 5.17 g/cm3) and volume (V, quasi-spheres with an average diameter of 50 nm) of the Fe3O4 SP core of the FMSPs, we can calculate the mass of each Fe3O4 SP core and the mass of iron in each FMSP. The number of FMSPs in each cell (nFMSPs cell) can be obtained by dividing the mass of iron in each cell by the mass of iron in each FMSP.
Immunofluorescence staining
For immunocytochemistry, after 4 h of FMSP incubation, PC12 cells were washed twice with PBS to remove free-floating FMSPs, fixed for 30 min with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 2% BSA for 1 h at room temperature. The cells were labeled with tubulin beta rabbit polyclonal antibody at 2 µg/ml in 0.1% BSA, incubated overnight at 4 °C, and then labeled with goat anti-rabbit IgG secondary antibody with Alexa Fluor 594 conjugate (Ex/Em: 590/617 nm), a dilution of 1:2000 for 2 h at room temperature. Nuclei were stained with DAPI (Ex/Em: 405/430-470 nm).
Cellular uptake kinetics
To study the effect of FMSP concentration on cellular uptake, PC12 cells were incubated with FMSPs at concentrations ranging from 5 to 20 μg/ml (5, 10, 15, 20 μg/ml). To study the effect of incubation time on the internalization of FMSPs, PC12 cells were incubated with FMSPs for 1, 2, 3, 4, 5 and 6 h. PC12 cells were seeded and incubated in 35 mm glass-bottom dishes (5×105 cells per dish) for fluorescence microscopy imaging or in 6-well plates (5×105 cells per well) for fluorescence activated cell sorter (FACS) analysis. A fluorescence microscope (IX51, Olympus Corporation, Tokyo, Japan) was used to examine the time- and concentration-dependent uptake, and the intracellular fluorescence intensity of FMSPs was qualitatively observed. For the FACS analysis (FACSAria II, Becton, Dickinson and Company, NJ, USA), the fluorescence intensity in cells was quantitatively measured with laser excitation at 561 nm and emission filtered at 582 nm, with a 15 nm band width; 10 000 events were collected for each sample.
In a separate experiment, energy-dependent uptake was performed with low-temperature incubation and ATP depletion treatments. PC12 cells were precooled at 4 °C or pretreated with 2-DG (50 mM for 45 min, creating an ATP-depleted environment by interfering with carbohydrate metabolism) at 37 °C, and then FMSPs were added and incubated for another 4 h. The intracellular fluorescence intensity of FMSPs was detected using fluorescence microscopy and FACS.
Cellular uptake pathways
Fluorescence microscopy, FACS and TEM were used to study the exact uptake mechanism of FMSPs. Different endocytosis inhibitors were used to determine which pathway was mainly involved in the uptake of FMSPs according to the description in a previous publication [22]. Two millimolar MβCD (depleting cholesterol from the cell membranes) and 50 μg/ml genistein (inhibitor of tyrosine protein kinase blocking the phosphorylation of caveolin-1) were used to inhibit caveolae-mediated endocytosis. Chlorpromazine (10 μg/ml, inhibiting clathrin-coated pit formation) was used to inhibit clathrin-mediated endocytosis. Aminoride (50 μM, interfering with membrane Na+/H+ATPase) and Cyto D (5 μM, inhibiting F-actin polymerization) were used to suppress micropinocytosis. PC12 cells were pretreated with inhibitors for 45 min, after which FMSPs (10 μg/ml) were added and coincubated for an additional 4 h prior to fluorescence microscopy and FACS analysis. Cells not incubated with FMSPs served as the negative control group, and a group treated only with FMSPs was used as the positive control.
Finally, TEM (EP 5018/40/Tecnai Spirit Biotwin 120 KV, FEI Czech Republic s.r.o, Holland) was used to study the successive stages of FMSP internalization. PC12 cells were incubated with FMSPs (10 μg/ml) for 4 h. Subsequently, cell samples were harvested and observed using TEM operating at 120 kV. Details of the specimen preparation are presented in Additional file 1: Supplementary Information.
Intracellular trafficking and distribution
To further determine the intracellular trafficking and distribution of FMSPs, we assessed the colocalization of FMSPs with cellular organelles by using confocal laser scanning microscopy (CLSM, FV3000, Olympus Corporation, Tokyo, Japan) images. We used cellular organelle-specific fluorescent probes, including LysoTracker Red (Ex/Em: 561/610-710 nm), Golgi-RFP (Ex/Em: 561/580-680 nm), ER-Tracker Red (Ex/Em: 561/610-710 nm) and MitoTracker Red (Ex/Em: 561/610-710 nm), to determine the intracellular distribution of FMSPs (Ex/Em: 488/500-580 nm) by assessing the colocalization of FMSPs with lysosomes, Golgi apparatus, endoplasmic reticulum, and mitochondria, respectively. The experimental process was performed according to the manufacturer's instructions (http://www.thermofisher.com). The images were captured with a 60× oil immersion objective with 3.2× magnification. Twenty different visual fields of each sample were analyzed, and triplicate experiments were performed. The colocalization of FMSPs with cellular organelles was analyzed by image analysis software ImageJ (http://rsb.info.nih.gov/ij/). Subsequently, as an additional validation experiment, TEM was used to directly observe the distribution and localization of FMSPs in the cellular ultrastructure.
Exocytosis of internalized FMSPs
The exocytosis of internalized FMSPs was studied using FACS. After 4 h of preincubation with FMSPs, the cells were washed with PBS three times and cultured in fresh culture medium without FMSPs. After further incubation for 2, 4, 8, 12 and 24 h, the cells were collected for FACS analysis to measure the intracellular mean fluorescence intensity.
Magnetic field preparation and quantification of magnetic forces
One perpetual cuboid neodymium magnet (NdFeB N48, cuboid side 50 mm×30 mm×10 mm) was applied to the right side of the culture dish to generate a gradient magnetic field to the cells at the center of the dish (Fig. 1a). The magnetic field was simulated by means of numerical field calculations using the software Comsol Multiphysics 4.3b (Comsol Multiphysics GmbH, Goettingen, Germany). A digital Gauss-meter (Scientific Equipment Roorkee, DGM-204) was used to measure the magnetic flux density induced by the setups of the neodymium magnet.
The exact explanation of the quantification of magnetic forces is as described in reference [6,23]. A magnetic particle within a magnetic flux density gradient (∇B) experiences magnetic forces F directed toward regions with higher field density due to its magnetic momentum (m):
[Please see the supplementary files section to view the equation.] Eq. (1)
In our experimental setup, the derivative of flux density B(T) along the magnetic field gradient inside the magnetic applicator is dB/dr (T/m). Superparamagnetic nanoparticles in gradient magnetic fields exert force due to a combination of parameters. As we find a value of FMSP saturation magnetization Ms, the density ρ and volume V, we can assume the net force FFMSP of FMSP:
[Please see the supplementary files section to view the equation.] Eq. (2)
The mass of iron taken up by PC12 cells was measured, and the number of FMSPs per cell (nFMSPs cell) was calculated. A single cell will thus be subject to a force Fcell given by FFMSP multiplied by the number of FMSPs in the cell:
[Please see the supplementary files section to view the equation.] Eq. (3)
Neurite oriented growth assay
PC12 cells were used to examine the magnetically guided outgrowth of neurites in directional orientation. PC12 cells were seeded in ibidi petri dishes. After 24 h of seeding, cells were treated with 10 μg/ml FMSPs and incubated again for 4 h to allow the FMSPs to interact with cells. The dishes were then placed inside the magnetic applicator. After 24 h, the outgrowth of neurites was induced under an external magnetic force, and the angle θ between the long axis of a neurite, the direction of the magnetic field (Fig. 1b), and the neurite length were measured. Neurite orientation was quantified as an orientation index (Oi), which was defined as Oi = cos(θ), with 0 <θ<π (when θ is 0, neurites with their long axis parallel to the magnetic field vector will have an Oi of +1, while when θ is π, neurites opposite to the magnetic field vector will have an Oi of -1. Randomly oriented neurites will have an average Oi of 0).
Four experimental groups were tested: (1) the treatment group, cells treated with both FMSPs and magnetic field (FMSPs+, M+), (2) the FMSP control group, treated with FMSPs and no magnetic field (FMSPs+, M−), (3) the magnetic field control group, without FMSPs and treated with a magnetic field (FMSPs−, M+), and (4) the blank control group, without FMSPs and with a null magnetic field (FMSPs−, M−). Experiments were performed in triplicate. For each experiment, 625 pictures were acquired under microscopic high-power fields (20× objective with 2.5× zoom) at the centers of the dishes. For each picture, all of the neurites were measured. Analysis was performed using ImageJ software.
Velocity measurement of DRG axonal elongation
To determine whether external magnetic force stimulation affects growth cone motility and the rates of axonal elongation in neurons, we used two-compartment microfluidic chambers for DRG neuronal culture. Previous studies have demonstrated that microfluidic chambers composed of two compartments (cell-body and distal-axon compartments) interconnected by microchannels (whose height is designed to be much smaller than the size of the cell body) can be used to separate axons from their cell bodies (Fig. 1c) [21,24-27].
Neurons were initially plated in the left cell-body (CB) compartments, and cells extend long axons (~1 μm diameter) that are smaller than the microchannels and grow across the microchannels into the right distal-axon (DA) compartments after a few days. The growth and extension of axons from CB compartments to DA compartments via microchannels can be used to simulate the process of bridging nerve defects by axons in vivo. We used time-lapse imaging to monitor the dynamic process of axon growth continuously, including the characterization and quantification of the underlying behavior of individual axons and growth cones. During time-lapse recording, cultures were recorded at 2.5 min/frame with differential interference contrast (DIC) optics (20× objective with 2.5× zoom). The time for the extending axons to grow across 150-µm-long microchannels was recorded. The average elongation rates of axons were then constructed from each individual axon growth trajectory. In this study, the average speed of axons crossing through the microchannels instead of their directionless extension is measured as the axonal elongation rate. Experiments were carried out in triplicate, and more than 40 trajectories from a single axon were collected and measured in each experiment.
mRNA transcriptome sequencing and bioinformatics analysis
To further determine the effects of mechanical signals mediated by FMSPs on the gene expression profile in neural cells, we also performed mRNA transcriptome sequencing and bioinformatics analysis on cell samples from different experimental groups. Experiments were carried out in triplicate.
RNA extraction, cDNA library establishment and Illumina sequencing: Total RNA was isolated from cultured sample cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the purity, concentration and integrity of RNA were checked and controlled. mRNA enrichment was achieved by the combination of oligo (dT)-attached magnetic beads and the poly A tail. The mRNA interrupted fragments were synthesized into a strand of cDNA with random hexamer primers, and then the buffer solution, dNTPs and DNA polymerase used to synthesize the second-strand cDNA. The purified cDNA was terminally repaired, ligated, and PCR amplified to obtain the final cDNA library. The Illumina HiSeqTM 2000 sequencing platform was used for sequencing. All sequence data were submitted to the NCBI database under the project accession number PRJNA597946 (https://www.ncbi.nlm.nih.gov/sra/PRJNA597946). To ensure the accuracy of subsequent bioinformatics, the original sequencing data were first filtered to obtain high-quality clean data. Then, using Hisat2 software (version 2.1.0, https://ccb.jhu.edu/software/hisat2/index.shtml) [28], sequencing reads were aligned to the rat reference genome (R. norvegicus, UCSC rn6; https://ccb.jhu.edu/software/hisat2/faq.shtml) to obtain high-quality sequencing data. Moreover, featureCounts software [29] (version 1.6.0) was used to analyze the gene expression level. The details of the quality control of the RNA extraction and the processing of raw sequencing data are presented in Additional file 1: Supplementary Information.
Screening differentially expressed mRNA: The obtained raw data were standardized with DESeq2 software (version 1.18.1, http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) [30], followed by pairwise comparison using the Wald test in DESeq2. Differentially expressed mRNAs were identified as those with p-values < 0.05 and |logFC| >1. Gene Ontology (GO) analysis was utilized to analyze the putative functions of key genes using clusterProfile in R (version 3.8.1, http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html)[31]. Upregulated differentially expressed mRNAs were subjected to GO biological process functional annotation [32] and enrichment analysis. A p-value less than 0.05 was used as the cut-off for the differential gene expression of significantly enriched GO terms.
Quantitative real-time PCR
Cell samples from different groups were washed three times with precooled PBS. Cell lysis was performed for total RNA extraction by adding 1 ml TRIzol reagent. The obtained RNA was reverse transcribed to prepare cDNA using PrimeScript RT Master MIX (Takara, Dalian, China), followed by PCR amplification. PCR was used to quantify differences in mRNA expression using the PowerUp SYBR™ Green Master Mix Kit (Waltham, MA, USA). GAPDH was applied as an internal control. The primers used in PCR were synthesized by Invitrogen (Shanghai, China), and the sequences are listed in Table S2 of Additional file 1: Supplementary Information. PCR conditions were as follows: initial denaturation at 95 °C for 10 min; 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 1 min, and amplification was performed for 40 cycles, followed by extension at 72 °C for 10 min. The multiple relationships of gene expression changes in different cells were calculated by using the 2-ΔΔCt method. Each reaction was performed three times.
Western blots
Cell samples from different groups were washed with PBS and subsequently resuspended in radioimmunoprecipitation assay (RIPA, Beyotime, Shanghai, China) lysis buffer. Cell lysates were then collected by centrifugation (12,000 rpm for 15 min at 4 °C). Total proteins were quantified using the Bicinchoninic Acid (BCA, Thermo, Scientific, California, USA) Protein Assay Kit. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). A solution of 5% nonfat dry milk was used to block the membranes for 1 h. Subsequently, the membranes were incubated with the primary antibodies rabbit anti-Cdh11 (1:10000), rabbit anti-Csf1r (1:10000), rabbit anti-Ppp1r1c (1:10000), and mouse monoclonal anti-β-actin (1:10000) overnight at 4 °C. After three washes in PBST buffer, membranes were incubated with anti-mouse antibody (1:5000) at 37 °C for 1 h and then washed three times in PBST. The special bands were visualized using an electrochemiluminescence (ECL) method (Millipore, Bedford, MA, USA). TanonImage software (Tanon Science & Technology, Shanghai, China) was used to conduct grayscale analysis for protein expression. Experiments were carried out in triplicate.
Statistical analysis
Continuous variables with normal distribution, such as cell viability, fluorescence intensity of cells, and elongation rates of axons, were represented as the mean ± standard deviation (SD). The distributions of the variables of neurite orientation index (Oi) and neurite length were found to be nonnormal by the Kolmogorov–Smirnov test; therefore, their values were represented by the median and interquartile range (Q1-Q3). Statistical significance was calculated using either one-way ANOVA (no rejection of normality) or nonparametric Kruskal-Wallis ANOVA (normality rejected). All of the analyses were conducted with SPSS (version 18.0, Chicago, IL, USA), and P<0.05 was considered to be statistically significant.