In vitro study
Isolation and culture of human bone marrow MSCs: hMSCs were isolated from the bone marrow of normal donors who had provided informed consent for the research project (CAAE - 27665714.4.0000.0071), which was approved by the ethics committee for research at the Instituto Israelita de Ensino e Pesquisa Albert Eisntein (São Paulo, Brazil).
The aspirated bone marrow was diluted with phosphate-buffered saline (PBS) (Gibco®, Carlsbad, CA, United States) (1:3) then centrifuged with 20 mL of Ficoll/Hypaque (GE Healthcare) for 30 min at 500 xg and 22 °C. Following centrifugation, the cells were removed from the plasma/Ficoll-Hypaque interface, washed 3 times with PBS, and resuspended in Dulbecco's modified Eagle's medium - high glucose (DMEM-HG) (Gibco®, Carlsbad, CA, United States) supplemented with 15% fetal bovine serum (FBS) (Gibco®, Carlsbad, CA, United States).
The hBM-MSCs were cultivated in 75 cm2 flasks with DMEM - low glucose (DMEM-LG) (GIBCO - Invitrogen Technologies, New York, USA), supplemented with 10% FBS, 1% de L-glutamine, 100 U/mL streptomycin and 100 U/mL penicillin (GIBCO - Invitrogen Technologies, New York, United States) and were maintained in humidified incubators with 5% CO2 at 37 ºC, to favor the attachment of cells to the flask bottom.
Immunophenotypic characterization of hBM-MSCs: Cell-surface expression was analyzed with a predefined set of protein markers. In brief, cells at the third passage with 70% confluency were stained with the selected monoclonal antibodies and incubated in the dark for 30 min at 4 ºC. The cells were then washed and fixed with 1% paraformaldehyde. The following positive human marker antibodies were used: CD29-PE (clone: MAR4; BD Pharmingen), CD44-PE (clone: 515; BD Pharmingen), CD73-PE (clone: AD2; BD Pharmingen), CD90-APC (clone: 5E10; BD Pharmingen), and CD105-PE (clone: 8E11; Chemicon, Temecula, CA, United States). The negative markers were as follows: CD14-FITC (clone: M5E2; BD Pharmingen, San Diego, CA, United States), CD19-APC (clone: SJ25C1; Biosciences), CD31-PE (clone: WM59; BD Pharmingen), CD34-PE (clone: 581; BD Pharmingen), CD45-PerCP-Cy5 (clone: 2D1; Biosciences, San Jose, CA, United States), CD106-FITC (clone: 51-10C9; BD Pharmingen), and human leukocyte antigen HLA-DR-PerCPCy5 (clone: L243; Biosciences). The cells were analyzed using FACSARia flow cytometry equipment (Becton Dickinson, San Jose, CA, United States), and the acquired data were analyzed using FLOWJO (Tree Star, Ashland, OR) software.
The hBM-MSCs were also subjected to differentiation induction to evaluate the multipotentiality characteristics and differentiation capacity of the cells into two cellular types: adipocytes and osteoblasts.
Lentiviral transduction of hBM-MSCs for BLI analysis: Cells were genetically engineered to generate luciferase-expressing hBM-MSCs (hBM-MSCLuc). Briefly, hBM-MSCs were transduced with the glycoprotein of the vesicular stomatitis virus (VSV-G) from pseudotyped viruses carrying the lentiviral vector (pMSCV_Luc2_T2A_Puro). The vector encodes the bioluminescent reporter luciferase-2 and the puromycin resistance gene puromycin N-acetyl-transferase under the control of a murine stem cell virus (MSCV) promoter.
For virion production, human embryonic kidney 293FT cells grown at 80% confluence in 150 mm Petri dishes (about 20 million cells/dish) were simultaneously transfected with 30 µg/dish of the vector pMSCV-Luc2-T2A-Puro along with two other helper vectors: 20 µg/dish of pCMV-dr8.91 and 10 µg/dish of pMD2.G. Transfection was conducted with 25-kDa linear polyethylenimine (PEI, Alfa Ansar) as previously reported. Two days after transfection, the viral supernatant was collected and filtered through 0.45 µm polyvinylidene fluoride (PVDF) filters and concentrated by ultracentrifugation. As described in previous reports, the copy number of integrated lentiviral vector sequences was determined via quantitative real-time polymerase chain reaction (PCR).
For lentiviral transduction, virions were added to cultures of 1 × 106 hBM-MSCs at a multiplicity of infection of 3 (MOI = 3) in the presence of 8 µg/mL polybrene (Sigma-Aldrich). The medium was replaced after 18 h, and the cells were cultured for an additional 48 h. After this period, the cells were selected for incubation with 1 µg/mL puromycin every other day for 8 d.
Bioluminescence signal expression in hBM-MSCLuc: The expression of the bioluminescence (BLI) signal in hBM-MSCLuc was analyzed in the following cell concentrations/well: 1 × 104, 1 × 105 and 1 × 106, in triplicate samples in a 24-well plate, using IVIS® Lumina LT Series III equipment (Xenogen Corp. CA, EUA). Images were captured before and after the addition of 100 μL of D-luciferin (50 mg/mL) (XenoLight, PerkinElmer), and the intensity of the BLI signal was detected under the following parameters: Exposure time of 2 ms with a 5 min interval between each image acquisition, over a total of 490 min. The kinetics of BLI expression were registered and analyzed with Living Image Software version 4.3.1 (IVIS Imaging System) in radiation absolute units (photons/s).
MNP with magnetic and fluorescent properties: We used multimodal nanoparticles (MNP-IR750; Molday ION™750 - BioPal) with an 8 nm iron oxide (Fe3O4) nucleus, a hydrodynamic size of 35 nm (coated with dextran), and a zeta potential of approximately +31 mV, which were conjugated with fluorophores that emitted fluorescence with of NIR absorption/emission wavelengths of 755/777 nm. The MNP-IR750 has magnetic and fluorescent properties detectable in MRI and NIR images.
MNP-IR750 characterization: Hydrodynamic size, zeta potential and optical properties: The hydrodynamic size and zeta potential of MNP-IR750 were measured using the dynamic light scattering (DLS) technique with the Zetasizer Nano S system (Malvern, United Kingdom). The hydrodynamic size distribution was obtained at an angle of 173º, with the number of averages set at 20 and a time of 5 s per mean. Measurements were performed in a fixed position at 25 ºC with a 60 s equilibrium period. In addition, to obtain information about possible agglomeration of nanoparticles, we performed an analysis of the stability of MNP-IR750 using DMEM-LG supplemented with 10% FBS over 18 h. The hydrodynamic size and zeta potential (surface charge) measurements were performed at a concentration of 50 µg Fe/mL and a pH of 7.4.
To verify the optical properties of MNP-IR750 excitation/emission, the corresponding spectrum was acquired using a Shimadzu RF-6000 fluorometer at a concentration of 100 µg Fe/mL, maintaining the temperature at 37 ºC.
hBM-MSCLuc labeled with MNP-IR750: For hBM-MSCLuc labeling with MNP-IR750, triplicate samples of 1 × 104 cells were placed in a 24-well plate in DMEM-LG, supplemented with 15% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL) and 1% L-glutamine. After 24 h of hBM-MSCLuc adhesion, the cells were washed twice with 300 µL of PBS and incubated for 18 h (at 37 ºC and 5% CO2), with MNP-IR750 added at the following concentrations: 5, 10, 20, 30, 40 and 50 µg Fe/mL, in DMEM-LG supplemented with 15% FBS. After incubation, the culture medium was removed, and the cells were washed three times with PBS.
Following the labeling of hBM-MSCLuc with MNP-IR750, the evaluation of MNP-IR750 internalized was performed via MRI, NIRF and BLI, and the viability of the labeled cells was assessed via the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and the BLI technique.
Internalization of MNP-IR750 into hBM-MSCLuc: Confirmation of the labeling of hBM-MSCLuc with MPN-IR750 was performed at the following concentrations: 5, 10, 20, 30, 40 and 50 µg/mL of MNP-IR750 in culture plates, via Prussian Blue staining[16,18]. Cells that were previously labeled and fixed in plates were washed twice with PBS and incubated with a staining solution composed of 0.25 mg of potassium ferrocyanide [K4Fe(CN)6] (SIGMA, United States) and 5% hydrochloric acid (HCl) in a proportion of 1:1, in a volume of 1000 µL per well over 5 min at room temperature, protected from light. After this period, the Prussian Blue solution was removed, and the wells containing hBM-MSCLuc were washed twice with 500 µL of PBS. Thereafter, 500 µl of Nuclear Fast Red staining solution (SIGMA, United States) was added to the wells, followed by incubation for 10 min. After staining, the cells were washed again with PBS (2x), and light-field images were obtained under a Nikon TI® inverted microscope.
MRI, NIRF and BLI signals after internalization of MNP-IR750 by hBM-MSCLuc: MRI, NIRF and BLI signals were evaluated in hBM-MSCLuc after labeling with MNP-IR750 at the following concentrations: 5, 10, 20, 30, 40 and 50 µg/mL.
For MRI signal evaluation, the labeled hBM-MSCLuc were mixed with 1% agarose (Sigma-Aldrich Chemie GmbH, Germany) and plated in culture wells. Images were acquired on a 3T RM scanner with a head coil of 32 channels (Magnetom Vision, Siemens, Germany) using the T2-weighted imaging sequence, following the protocol described in a previous study.
NIRF and BLI images were acquired after the plating of labeled hBM-MSCLuc. The BLI signal was acquired after the addition of 100 μL of luciferin (1 mmol/L in PBS), the maximum time of intensity of the BLI signal determined via kinetic BLI analysis (section 2.4), using an exposure time of 2 ms, binning of 2 and f/stop of 4. The NIRF signal was acquired in the same samples used for BLI, applying an excitation of 745 nm, registered in a range of emission of 810-875 nm. Both imaging analyses were performed with IVIS® Lumina LT Series III equipment, and the signals were analyzed in radiation absolute units (photons/s). The experiments were performed with hBM-MSCLuc between the fifth and seventh passages.
Viability of hBM-MSCLuc labeled with MNP-IR750 - MTT and BLI: Evaluation of the viability of hBM-MSCLuc labeled with MNP-IR750 was performed using the MTT and BLI assays.
In the MTT assay, hBM-MSCLuc were grown in 96-well plates until they were subconfluent. MPN-IR750 were then added to the cells at defined concentrations of 5, 10, 20, 30, 40 and 50 μg Fe/mL, followed by incubation overnight. After incubation, the culture medium was discarded, and 100 μL of fresh medium per well was added to the cells, after thorough washing with PBS. Then, 100 μL of the MTT reagent (1 mg/mL - final concentration) was added per well, and the plate was incubated for four h in an incubator, at 37 ºC in 5% CO2. Actinomycin D (Sigma-Aldrich) was used as positive control for cell death in this assay. The “cell death dose” concentration identified in this assay was 0.25 µg/mL. After incubation, the medium was discarded from the wells, and 100 µL of dimethyl sulphoxide (DMSO Hybri-Max - Sigma-Aldrich) was added to solubilize the formazan crystals that had formed. Readings were then taken in a DTX 880 Multimode Detector reader (Beckman Coulter) at 490 nm, with subtraction for plate absorbance at 650 nm. The viability percentage of the cells was calculated as the ratio of the mean absorbance of triplicate readings with respect to the mean absorbance of control wells, as cell viability = (sample/control) × 100.
The viability assays were verified using the BLI technique. Similar samples to those used in the MTT assays were used for the BLI assay, adding 100 mL of luciferin in each well, and acquiring the BLI images using IVIS® Lumina LT Series III equipment. For BLI intensity analysis (photons/s), a region of interest (ROI) of 2.5 cm2 was selected. The viability percentage was calculated with the formula (sample/control) × 100.
Quantification of MNP-IR750 internalized into hBM-MSCLuc: Quantification of MNP-IR750 after hBM-MSCLuc labeling was performed via MRI, ICP-MS and NIR imaging. The samples used for quantification were prepared with 1 × 106 hBM-MSCLuc that either were not labeled (control) or were labeled with MNP-IR750 at the following concentrations: 5, 10, 20, 30, 40 and 50 µg Fe/mL.
Quantification of MNP-IR750 internalized into hBM-MSCLucvia MRI. For quantification of the internalization process by MRI, the following equation was used:
(1) 1/( T2hBM-MSCLuc+MNP_IR750) = 1/( T2hBM-MSCLuc) + [Fe] × r2
where [Fe] is the concentration of intracellular iron internalized into hBM-MSCLuc; r2 is the relaxivity of the MNP-IR750; and T2 is the transverse relaxation time for samples containing hBM-MSCLuc labeled with MNP-IR750 and control samples (hBM-MSCLuc).
For the calculation of r2, a phantom with 24 wells (culture plate) containing MNP-IR750 suspended in the following concentrations was used: 0, 2, 4, 6, 10, 15 and 20 µg Fe/mL, dispersed in 1% agarose. The phantom was subjected to MRI examination and the T2 values of samples were determined from the relaxivity curves. The r2 values were obtained via linear adjustment of the inverse of the transverse relaxation vs the concentration of MNP-IR750 used for cellular labeling.
For the calculation of T2hBM-MSCLuc+MNP_IR750, samples labeled with different concentrations of nanoparticles, as described above, were used; for the determination of T2hBM-MSCLuc, only 1 × 106 hBM-MSCLuc from the samples were used. In both analyses, the samples were suspended in a 1% agarose solution and plated in a 24-well culture plate, then subjected to MRI examination.
T2-weighted MRI images were acquired in a whole-body 3T scanner (Magneton Vision®, Siemens, Erlangen, Germany) with a 32-channel head coil using the following parameters: Multicontrast turbo-spin echo sequence, repetition time (TR) of 1500 ms, echo time (TE) of 8-256 ms, field of view of 300 mm, 256 × 256 matrix, slice thickness of 3.0 mm and flip angle of 180°. The intensity curves of the MRI signals of the samples as a function of TE were analyzed using a selection of regions of interest with a fixed size. The T2 of each sample was determined by adjusting the decay curve with a monoexponential linear algorithm: Intensity (TE) = C × e(-TE/T2).
From the MNP-IR750 load obtained from equation 1, the number of MNP-IR750 internalized into hBM-MSCLuc was calculated with the following equation:
(2) Number of MNP_IR750 = [6 × loadMNP_IR750 × (at_m)]/[π × ρMNP_IR750 × MFe × φ3MNP_IR750]
Where loadMNP_IR750 is the internalization MNP-IR750 loaded into hBM-MSCLuc; at_m is the atomic mass; ρMNP_IR750 is the iron oxide density (Fe3O4); MFe is the molecular weight of iron; and φMNP_IR750 is the diameter of MNP-IR750.
Quantification of MNP-IR750 internalized into hBM-MSCLucvia ICP-MS. The samples were diluted in 1 mL of Milli-Q® water (EMD Millipore Corporation, Bedford MA, USA) and subjected to the digestion of organic components with 5 mL of nitric acid (37%) using a Titan Microwave sample preparation system (Perkin Elmer, USA). After digestion, the samples were analyzed with ICP-MS equipment (Perkin Elmer Nexion 350x, PerkinElmer Corporation, USA) to determine the iron content of each sample. Measurements of samples were performed in triplicate, and quantification was based on a calibration curve using certified standard iron (NexION # N8145054) at the following concentrations: 0, 10, 20, 30, 40 and 50 ng Fe/mL (ppb). Samples of 1 × 106 hBM-MSCLuc without labeling were used as a control.
Quantification of MNP-IR750 internalized into hBM-MSCLucvia NIRF imaging. NIRF images were acquired after trypsinization of the samples and washing with PBS. The NIRF signal was detected after excitation at 745 nm and was registered in the emission range of 810-875 nm using IVIS® Lumina LT Series III equipment. The absolute quantification was determined after establishing the calibration curve using known concentrations of 1, 2, 3, 4, 5 and 6 µg Fe/mL.
In vivo study
Animals and experimental design: We used 2-month-old male Wistar rats weighing 250-300 g. The animals were maintained at the vivarium of the Experimental Surgical Training Center (Centro de Experimentação e Treinamento em Cirurgia - CETEC) at 21 ± 2°C and 60% ± 5% relative humidity with full ventilation under a 12 h light/dark cycle (7 a.m. - 7 p.m.), and they had access to food and water ad libitum. This vivarium is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), and the general conditions were monitored daily. The study was approved by the Ethics in Animal Research Committee of the Hospital Israelita Albert Einstein (HIAE) with approval number 1906-13
The experimental design of the in vivo study involved in two experiments:
Experiment 1 was conducted to analyze the sensitivity of the NIRF, BLI and MRI signals of the labeled hBM-MSCLuc implanted at different concentrations in the animals in the sham group, after being subjected to a craniectomy procedure. These animals were divided into 4 groups (n = 7 rats/group): Group Sham_control (S_control) - implantation of 1 × 106 de hBM-MSCLuc; Group Sham_5 - implantation of 1 × 106 de hBM-MSCLuc labeled with 5 µg Fe/mL of MPN-IR750; Group Sham_20 implantation of 1 × 106 de hBM-MSCLuc labeled with 20 µg Fe/mL of MPN-IR750; and Group Sham_50 implantation of 1 × 106 de hBM-MSCLuc labeled with 50 µg Fe/mL of MPN-IR750.
Experiment 2 analyzed the NIRF, BLI and MRI signals of the labeled hBM-MSCLuc implanted in the animals after being subjected to stroke induction. These animals were divided into 2 groups (n = 7 rats/group): Group Sham_50: implantation of 1 × 106 de hBM-MSCLuc labeled with 50 µg Fe/mL of MPN-IR750; and Group Stroke_50: implantation of 1 × 106 de hBM-MSCLuc labeled with MPN-IR750. The concentration of nanoparticles that was used was determined from the best conditions verified in experiment 1.
In both experiments, the animals were randomly allocated, coded and housed in individual cages.
Evaluation of NIRF, BLI and MRI signals after the implantation of hBM-MSCLuc labeled with MNP-IR750 in animals (Experiment 1): A total of 28 Wistar rats were used to evaluate the behavior of the signals of the labeled and unlabeled hBM-MSCLuc in the animals’ brains. The animals were anesthetized with ketamine hydrochloride (100 mg/kg) and xylazine hydrochloride (20 mg/kg) i.p. and subjected to a craniectomy procedure to implant the cells at the following coordinates: 2.0 mm antero-posterior, 2.0 mm lateral to the midline and 2.5 mm deep, according to the atlas of Paxinos and Watson (1986). The cells were infused at a rate of 10 µL/min using a 10 µL Hamilton syringe.
After 4 h of cell implantation, the animals were subjected to evaluation of BLI expression and NIRF detection in vivo using IVIS® Lumina LT Series III equipment. The NIRF signal measurements were obtained with an excitation wavelength of 745 nm and an emission wavelength of 810-875 nm. Soon thereafter, the animals received 150 mg/kg of luciferin i.p., and the BLI images were acquired with 10 min of latency. Both images were analyzed in radiation absolute units (photons/s).
The animals were euthanized after in vivo NIRF and BLI evaluation. Their brains were extracted to record NIRF emissions using the same parameters employed for in vivo image acquisition. Following ex vivo NIRF analysis, the brains were fixed with 4% paraformaldehyde, and brain phantoms were prepared with 1% agarose for MRI signal evaluation. The phantoms with brain tissue were analyzed by using a 3T scanner (Siemens), to track the hBM-MSCLuc labeled with MNP-IR750. The MRI images were obtained with a 3D Fast Low Angle Shot (FLASH) sequence, a matrix of 256x160x128, TR = 200 ms, TE = 20 ms, and a range of excitation angle of 20-25º.
Induction of a focal ischemic lesion via thermocoagulation (stroke): A focal brain ischemic lesion was induced via thermocoagulation in the pial blood vessels of the motor and somatosensory cortex as previously described. Briefly, animals were anesthetized with ketamine hydrochloride (100 mg/kg, i.p.) and xylazine hydrochloride (20 mg/kg, i.p.) and placed in a stereotaxic apparatus (Harvard Apparatus, Holliston, United States). A craniectomy procedure was performed to expose the left somatosensory cortex (+ 2 to -6 mm in the anterior-posterior direction and +2 mm on the medial-lateral axis from the Bregma, according to the atlas of Paxinos and Watson). Superficial blood vessels were transdurally thermocoagulated by approximation of a hot probe to the dura matter (about 2 mm), maintaining a constant temperature of 400ºC for 30 min. The procedure was concluded with incision tissue suturing and the administration of a tramadol analgesic (5 mg/kg) (i.p.). Throughout anesthesia, the rats were placed on a heating pad to maintain the rectal temperature at 37.0 ± 0.5 ºC (PhysioSuite, kent Scientific Corporation, Torrington, CT, United States).
The ischemic lesion was confirmed through local blood perfusion analysis using a PeriCam Perfusion Speckle Imager (PSI) system (Perimed, Stockholm, Sweden) and TTC staining after 2 h of lesion induction, as described in a previous study. The color changes of the targeted region, from light red to dark red, were also noted.
Implantation of hBM-MSCLuc labeled with MNP-IR750 and evaluation of the signal in the brains of animals after focal ischemic lesion induction (Experiment 2): In experiment 2, after 24 hs of focal brain ischemic induction, the animals were subjected to cell implantation in the same manner described above in section 2.13. Then, 6 d after stroke induction, we analyzed the signal behavior of the hBM-MSCLuc labeled with MNP-IR750 via BLI and NIRF imaging in vivo and MRI ex vivo, following the same procedures described in section 2.13. The concentration of MNP-IR750 implanted in the stroke group was determined from the best result for the signal detected in the presence of the different concentrations tested in experiment 1.