Materials
Iron acetylacetonate, TREG, Diethylene Glycol and PEG were purchased from Aladdin Industrial Corporation. (shanghai, China). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich Corporation. (Shanghai, China). Dulbecco's modified eagle medium (DMEM), trypsin-EDTA (0.25%) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-Hydroxy succinimide were purchased from Sigma-Aldrich (China). N, N-Dimethylformamide, ethyl alcohol absolute and ethyl acetate were purchased from Sinopharm Chemical Reagent Corporation. Ultrapure water was produced by millipore pure and ultrapure water purification systems. Anti-Human CD90-FITC, Anti-Human CD45-PE, Anti-Human CD34-FITC and Anti-Human CD105-PE were purchased from Biolegend.
Synthesis of USPIO
The USPIO were synthesized by polyol method as previous research. [11–14] Steps of experiment as follow: iron acetylacetonate 1 mmol and TREG 25 mL were mixed in a three-neck flask, which connected to argon, spherical condensing tube and thermometer. After incubated with argon flow for 15 minutes, the mixture was heated to 120℃ (heating rate is 3℃/min) and maintained for 1 hours, then the mixture was heated further to 250℃ at the same heating rate and maintained for 30 minutes. The reaction mixture was natural cooled to room temperature, diluted with 2 mL absolute ethyl alcohol, followed by precipitation with ethyl acetate. The sediment was collected by centrifugation at 8000 rpm for 20 minutes, then resuspend in absolute ethyl alcohol. The USPIO were stored in absolute ethyl alcohol at 4℃ for further use.
Preparation of PAA/PEG coated USPIO
The USPIO-PAA was acquired according to a previous report [15]. In detail, polyacrylic acid (PAA) 1.5 g was dissolved into 24 mL diethylene glycol. After a 5 minutes argon flow, the mixture was heated to 110℃ and maintained until the solution pellucid. The ethanol dispersing solution containing 28 mg of USPIO prepared in the previous step was added to the above clarified solution after ultrasonic dispersing. The solution was finally heated to 210℃ at a rate of 3℃/min. The reaction was stopped after 2 hours of reflux and naturally cooled to room temperature. After cooling, ethyl acetate was added to the reaction solution to precipitate the USPIO NPs, and then centrifuged at 8000 rpm for 20 min to remove the supernatant. The precipitate was then dispersed in ethanol, followed by precipitation with ethyl acetate. After the washing procedures for three times, the USPIO-PAA was dispersed in ultrapure water for further use. In order to improve biocompatibility, we further linked PEG to the surface of nanoparticles by EDC and NHS-mediated amidation [16]. 0.45 g of PEG was dissolved in 5 ml ultrapure water, and added to the 15 ml USPIO-PAA dispersion (contains 35 mg Fe) supplemented with 20 mg EDC and 12 mg NHS. The mixture was mechanical stirring at room temperature for 2 h, then 10 ml DMF solvent was added, removing water at 40℃ in the rotary evaporation apparatus. Finally, 40 mg DEC and 24 mg NHS were added to the mixture, mixing at room temperature for 48 h. The reaction solution was transferred to a dialysis bag in ultrapure water for 72 h, during which ultrapure water was replaced every 12 hours. The mixture was further transferred into an ultrafiltration tube to collect molecules with an Mr > 30kD by centrifugation at 4000 rpm for 10 minutes. The final product was dissolved in ultra-pure water and stored at 4℃.
Characterization of NPs
The particle size, size distribution and morphology of the samples were analyzed by TEM. The coating layer was measured by negative stained. After ultrasonic dispersion, the USPIO-PAA and USPIO-PAA/PEG in aqueous solution were added to the copper network of 300-mesh carbon supporting membrane, dried naturally, and then put into a projection electron microscope (Tecnai G2F20 s-twin, FEI) for observation.
The infrared spectroscopy analysis is performed on Agilent Technologies Cary 600 Series FTIR Spectrometer, where the dried sample powder is added directly to the sample tank for detection.
The Fe content in USPIO-PAA/PEG dispersion was determined by flame atomic absorption spectrometry (FAAS), whose model is Perkin Elmer Analyst 700. First, 1, 2, 3, 4, and 5 µg/mL iron standard solution was configured to draw the standard curve, then 100 µL USPIO-PAA or USPIO-PAA/PEG dispersion solution was dissolved with nitric acid, and the content of iron was determined in a 50 mL volumetric flask.
Magnetic resonance imaging of the nanoparticles was conducted on clinical scanners with magnetic field of 3 T in the imaging department of the first affiliated hospital of Soochow University. The nanoparticles were dispersed in 1% agarose gel solution according to the corresponding concentration. After the solution solidified, the transverse relaxation time of the samples was measured by multi-echo sequence. Exporting the acquisition of MRI in DICOM format, and then use the RadiAnt DICOM Viewer to open, read the grey value, by importing the gray values of different echo times into Origin for fitting, the transverse relaxation time values of USPIO dispersions with different concentrations were obtained, finally, the transverse relaxation rate r2 of USPIO-PAA and USPIO-PAA/PEG samples was obtained by linear fitting with the transverse relaxation rate of 1/T2 and the Fe concentration of the samples.
HADSCs Isolation and Identification
All studies were done in accordance with the ‘Ethical Guiding Principles on Human Embryonic Stem Cell Research’ (of the Ministry of Science and Technology and the Ministry of Health, People’s Republic of China, 2003) and Helsinki Declaration. Adipose samples were obtained with informed consent and ethical approval from the first affiliated hospital of Soochow University. The adipose tissue was washed, cut into small pieces after removing the blood vessels and connective tissue under an anatomical microscope. The blocks were digested with type I collagenase (0.3 pzu/mL) at 37℃ for 30 min, followed by centrifugation at 500 g for 10 min. The cell pellet was suspended with in DMEM + 10% FBS and seeded at the density of 8 × 104/cm2. After 48 hours, the old medium containing floating cells was discarded and replaced with fresh medium.
HADSCs were characterized by flow cytometry for surface markers specifically labeling mesenchymal (CD90 and CD105) and hematopoietic (CD34 and CD45) stem cells. A total of 1 × 105 cells were harvested and incubated with either PE, FITC, APC/cy7 or PerCP conjugated antibodies against CD34, CD45, CD90and CD105 mouse anti-human monoclonal antibodies and appropriate isotype controls. Stained cells were analyzed using a flow cytometer (LSRFortessa, BD, USA), and data were analyzed using FlowJo software. Four phenotypes of CD90+, CD105+, CD34- and CD45- were selected for the surface markers of HADSCs. Expression levels of cell surface markers were identified by flow cytometry (BD LSRFortessa).
The adipogenic and osteogenic differentiation of HADSCs were evaluated by Oil Red O and Alizarin Red Staining, respectively. HADSCs were cultured with either MesenCultTM Adipogenic Differenation medium (Stem cell Tech., 05412) or OriCellTM Osteogenic Differentiation Kit (Cyagen, HUXMA-90021). For adipogenic differentiation of HADSCs, cells were assessed on day 14 by qualitative Oil Red O staining for lipid-filled mature adipocytes (VivaCell Biosciences, C37A00150). For osteogenic differentiation of HADSCs, cells were assessed on day 21 by Alizarin Red Staining for calcium nodule in mature osteocytes (VivaCell Biosciences, C37C00150). Images were acquired using an inverted Nexcope microscope.
In vitro cellular uptake of USPIO-PAA/PEG and Biocompatibility evaluation
HADSCS were plated in a 6-well plate with a density of 1 × 106 per well. Cells were incubated with the medium containing USPIO-PAA/PEG (Fe 10 µg/mL) for 2 hs, and stained with Prussian blue staining for intracellular Fe identification.
The biocompatibility and cytotoxicity of USPIO-PAA/PEG was determined by MTT colorimetry. HADSCS were plated in a 96-well plate at a density of 5 × 103 per well, incubated with different concentrations (0、10、20、40、80、160) of USPIO-PAA/PEG 24 h, 48 h or 72 h. 20 µl MTT solution (5 mg/mL) was added to each well for 4 h, followed by 150 µL dimethyl sulfoxide to dissolve the crystals. Absorbance value of each hole was measured at OD 570 nm using enzyme-labeled instrument. (BioTek Synergy HT)
6-OHDA induced PD Model
15–20 of male Wistar rats (SPF grade, weighing 220 ± 20 g) were used for in vivo tracing of USPIO-PAA/PEG-labeled HADSCs on PD animals. All procedures were performed according to the Regulations in China (Regulations for the administration of affairs concerning experimental animals, 2017) and approved by the Institutional Animal Care and Use committee at Chinese Academy of Sciences. Throughout, animals were housed under controlled illumination (12/12-hour light/dark cycle, “on” at 7am) with ad libitum access to food and water. PD model was prepared by 2-point injection of 6-hydroxydopamine (6-OHDA,Sigma, St. Louis, MO, USA) into the unilateral striatum of rats. As described previously [17, 18], rats were stereotaxically injected with 3 µL of 6-OHDA solution (5 µg/µL) at 2 coordinates (AP:1.2 mm, ML:2.2 mm, DV:-4.0-6.0 mm; and AP:-1.0 mm, ML:4.4 mm, DV:-4.5-6.5 mm) respectively. Apomorphine-induced rotation (0.5 mg/kg, subcutaneously) tests were used to test the validity of PD models. The rats were injected with apomorphine (0.5 mg/kg) subcutaneously at 1, 2 and 3 weeks following 6-OHDA treatment, and the rotation scores were evaluated for 30 min in an open field. The PD model has more than 7 roations per min induced by apomorphine was considered succeed.
Cell transplantation and in vivo MRI imaging
HADCs were incubated with USPIO-PAA/PEG(iron concentration was 10 µg/mL) for 2 hs when they reached 80% confluence. 3 weeks after PD model preparation, 3 × 106 of USPIO-PAA/PEG –labeled HADCs or saline were injected into the left striatum of PD rat models at the following coordinate (AP:1.2 mm, ML:2.2 mm, DV:-4.0-6.0 mm). These animals were subjected to the apomorphine-induced rotation tests at 1, 2 or 3 weeks after the transplantation surgery.
For in vivo MRI, animals were imaged using an In vivo imaging system (IVIS) small animal imaging system (Perkin Elmer, Waltham, MA, USA) at the 3th, 9th, 15th and 21th following the saline or HADSCs injection.
Histology analysis
The brains of the remaining rat models were harvested and sectioned to a thickness of 30 mm for analysis 3 weeks post-stem cell transplantation (n = 5 per group). Sections were incubated with anti-tyrosine hydroxylase (TH, abcam, England), and visualized with Alexa-594-conjugated donkey anti rabbit antibody (Abcam). DAPI was used for counterstaing with nuclei. Images were captured using Leica TCS SP5 confocal microscope.
Statistical Analysis
Numerical data were expressed as the mean ± SD. Data were subjected to 2-tailed Student t tests or one-way ANOVA using GraphPad Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA) to evaluate the difference. * indicates p < 0.05 and NS indicates no significant difference.