D-mannose-Coating of Maghemite Nanoparticles Improved Labeling of Neural Stem Cells and Allowed Their Visualization by ex vivo MRI after Transplantation in the Mouse Brain

Synthesis and Characterization of Nanoparticles

The D-mannose-modified/coated maghemite nanoparticles (D-mannose(γ-Fe₂O₃)) and unmodified/uncoated maghemite nanoparticles (Uncoated(γ-Fe₂O₃)) were prepared by in situ precipitation of iron oxide in D-mannose solution method as described previously ⁴⁷ . Briefly, γ-Fe₂O₃ nanoparticles were obtained by chemical co-precipitation of FeCl₂ and FeCl₃, followed by oxidation of the produced magnetite with sodium hypochlorite to maghemite (γ-Fe₂O₃). γ-Fe₂O₃ nanoparticles were coated post-synthesis with D-mannose ⁴⁵ . Detailed examination and characterization of the nanoparticles after synthesis was done by transmission electron microscopy (TEM) as described previously ^45,48,49 . Briefly, the morphology of the particles was evaluated at 120 kV using a Tecnai Spirit G2 transmission electron microscope (FEI, Brno, Czech Republic) and the micrographs processed by NIS Elements image analysis program (Laboratory Imaging, Prague, Czech Republic).

Animals

The mouse inbred strain C57Bl/6NCrl was used. The animals were housed in a temperature (22 ± 2°C) and humidity controlled environment, under 12/12 hours light/dark cycles. Water and pelleted food were given ad libitum. All animal procedures were approved by the internal review board of the ethics committee of the School of Medicine University of Zagreb and were in accordance with the ethical codex of the Croatian Society for Laboratory Animal Science and with EU Directive 2010/63/EU on the protection of animals used for scientific purposes.

Neural Stem Cell Culture and Treatment

Neural stem cells were isolated from mouse fetuses at gestational day 14.5 (E14.5) as described previously ^{50 –52} . Briefly, pregnant females were sacrificed and neural stem cells were isolated from the telencephalic wall of E14.5 fetuses by microdissection and dissociation using StemPro Accutase (Gibco by Thermo Fisher Scientific, Waltham, MA, USA). Individual neural stem cells were obtained by trituration. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂/95% O₂. Expansion medium contained: DMEM/F-12 with GlutaMAX (Gibco by Life Technologies), 1% N2 Supplement (Gibco by Life Technologies), 2% B27 supplement (Gibco by Life Technologies), 1% penicillin/streptomycin (Gibco by Life Technologies), recombinant mouse epidermal growth factor (EGF) 20 ng/ml (Gibco by Life Technologies) and recombinant mouse basic fibroblast growth factor (bFGF) 10 ng/ml (Gibco by Life Technologies). Cells were cultivated in suspension. After 2 days neurospheres were formed. Neurospheres were dissociated using StemPro Accutase (Gibco by Thermo Fisher Scientific, Waltham, MA, USA), plated on 6-well plates at 2×10⁵ NSC/well, and allowed to attach for 24 h for Prussian blue staining, TEM, and flow cytometry experiments. All plates were previously coated for 12 h with 50 µg/ml poly-D-lysine water solution (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). For intracerebral transplantation purpose, neurospheres were dissociated by StemPro Accutase and 200,000 cells were transplanted in 1 μl of cell medium. Neurospheres were dissociated with StemPro Accutase and plated on 24-well plates at a cell density of 4×10⁴ NSC/well for MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium bromide) cell viability assay. Cells were cultivated as free-floating aggregates in suspension for neurosphere assay purpose. Neurospheres were dissociated, single cells and were plated on uncoated 6-well plates (250,000 cells per well), and were allowed to develop into neurospheres in a humidified atmosphere with 5% CO₂ at 37°C.

Neurospheres were first dissociated using StemPro Accutase, single cells and small neurospheres were plated on 12 mm coverslips (250,000 cells per coverslip) previously coated with Poly-d-lysine (500 μg/ml, 24 h at 37°C, Sigma-Aldrich) and laminin (10 μg/ml, 24 h at 37°C, Sigma-Aldrich) for the purpose of differentiation analyses. To the cells used for in vitro proliferation experiments, Uncoated(γ-Fe₂O₃) or D-mannose(γ-Fe₂O₃) nanoparticles were added for 48 h and left to proliferate for an additional 48 h in a medium with proliferation factors. Cells were fixed with 4% paraformaldehyde (PFA) (pH 7.4) on the 5th day after plating. For in vitro differentiation experiments Uncoated(γ-Fe₂O₃) or D-mannose(γ-Fe₂O₃) nanoparticles were added for 48 h to cells left to proliferate for an additional 5 days in a medium without proliferation factors. Cells were fixed with 4% PFA (pH 7.4) on the 8th day after plating.

D-mannose(γ-Fe₂O₃) and Uncoated(γ-Fe₂O₃) nanoparticles were added directly to the culture medium 24 h after NSC plating and incubated for 48 h. D-mannose(γ-Fe₂O₃) and Uncoated(γ-Fe₂O₃) nanoparticles were used in the following concentrations: 0.002, 0.01, 0.02, 0.03, 0.04, 0.1, 0.15, and 0.2 mg/ml. The nanoparticles were not added to the control unlabeled cells.

To determine the mechanism of nanoparticle uptake, after seeding and attaching NSCs were pre-treated with various inhibitors of endocytosis for 30 min and then incubated with 0.2 mg/ml of D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles for the next 48 h in the presence of the inhibitor ⁵³ . The inhibitors used were: phenyl arsine oxide (12 nM, Sigma-Aldrich), cytochalasin D (60 nM, Sigma-Aldrich), nocodazole (20 nM, Sigma-Aldrich), and filipin (0.3 µg/ml, Sigma-Aldrich).

Prussian Blue Staining

After a 48 h incubation period the medium with nanoparticles was removed, cells were washed three times with phosphate-buffered saline (PBS), fixed with 4% PFA (Sigma-Aldrich) for 20 min and stained with 1:1 mixture of 10% K₄Fe(CN)₆ (Sigma-Aldrich) and 20% HCl for 20 min. Cells were counterstained with 0.1% Nuclear Fast Red (Sigma-Aldrich) for 1 min, mounted with HistoMount (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) and coverslipped. After drying, the cells were analyzed under bright field using an ECLIPSE E200 light microscope (Nikon Instruments, Tokyo, Japan).

Flow Cytometry

After labeling, the cells were dissociated with StemPro Accutase (Life Technologies), washed once with PBS, resuspended in PBS containing 2% FBS and 2 mM EDTA (pH 7.4) and passed through a 40 µm Falcon™ cell strainer (Fisher Scientific by Thermo Fisher Scientific, Waltham, MA, USA). To determine the percentage of cells labeled with nanoparticles, the increase of the side scattered (SSC) light of the laser beam was measured using the Attune® Acoustic Focusing Flow Cytometer (Applied Biosystems, Foster City, CA, USA). The intensity of the SSC is proportional to the intracellular density ⁵⁴ . The percentage of positive cells was determined with FCS Express 4 software (De Novo Software, Glendale, CA, USA) using the Overton cumulative histogram subtraction method ⁵⁵ .

The effects of inhibitors on cellular uptake of nanoparticles were examined using Attune® Acoustic Focusing Flow Cytometer and FlowJo vX.0.7 software (Tree Star, Inc., Ashland, OR, USA).

Transmission Electron Microscopy

The cells treated by nanoparticles were detached from the surface by cell dissociation reagent StemPro Accutase (Life Technologies), washed once with DMEM/F-12 medium, shortly centrifuged and fixed overnight with 2% glutaraldehyde. The fixed cells were washed 3 times 15 min each with 0.1 M phosphate buffer (PB), post-fixed in 1% osmium tetroxide in 0.1 M PB for 1 h, washed 3 times 15 min each with 0.1 M PB and rinsed with water for 10 min. After rinsing, NSCs were immersed in 2% uranyl acetate in water for 1 h, then dehydrated in graded series of ethanol (20%, 50%, 70%, 90%, 15 min each), followed by two 100% ethanol washes, and two 15 min acetone washes. After each step the cells were centrifuged for 1 min at 1,500 g to settle, the supernatant removed, and the solution changed. For embedding in the Durcopan (Merck KGaA, Darmstadt, Germany) the cells were placed in 1:1 mixture of acetone/Durcupan resin for 3 h at room temperature, after which they were transferred to 100% Durcupan resin, for 72 h polymerization at 64°C.

Using a diamond knife (DiATOME) on an ultramicrotome RMC Power Tome XL (Boeckeler Instruments, Tucson, AZ, USA) semi-thin sections were cut and stained with 0.2% toluidine blue solution (Sigma-Aldrich). Sections were examined under a light microscope (ECLIPSE E200, Nikon Instruments). Subsequently, from selected samples 70 nm ultra-thin sections were cut, picked up on copper grids, and contrasted with 2% uranyl acetate (Merck) and Reynolds lead citrate. The sections were examined using a TEM902A transmission electron microscope (Zeiss, Oberkochen, Germany) operated at 80 kV, using magnifications ranging from 7,000 to 30,000.

Stereotaxic Transplantation of Neural Stem Cells Into the Mouse Brain

Together with nanoparticle labeling, the cells were treated just prior to transplantation with PKH26 fluorescent dye (PKH26 Red Fluorescent Cell Linker Kit for general cell membrane labeling, Sigma-Aldrich) following the manufacturer’s instructions. NSC were dispersed and resuspended in Hank's balanced salt solution (HBSS, Invitrogen). Animals were anesthetized with an intraperitoneal injection of Avertin (Sigma-Aldrich) at a dose of 0.5 g/kg and fixed in a stereotactic frame (KOPF stereotaxic apparatus 900LS). After exposing the skull by a small incision, a hole was drilled at the following coordinates (in mm) relative to bregma: anteroposterior −1.3, mediolateral +2.0 and dorsoventral −1.5 (from dura), determined according to the stereotaxic atlas ⁵⁶ . We injected 2 μl of homogeneous cell suspension in HBSS buffer containing 400,000 of cells into the brain striatum through a Hamilton syringe needle, which was kept in place for 5 min before being slowly retracted. The wound was closed with silk suture and the animals were kept for an hour on a heating pad to recover prior to returning to their cages.

Mice were anesthetized using Avertin (0.5 g/kg) 72 hours after NSC transplantation and subsequently transcardially perfused with freshly prepared PB (0.1 M, pH 7.4) followed by buffered 4% PFA (in 0.1 M PB, pH 7.4). Brains were carefully dissected and post-fixed by immersion in the same fixative at 4°C overnight.

Magnetic Resonance Imaging

To validate the MRI visibility of the analyzed D-mannose(γ-Fe₂O₃) nanoparticles in ex vivo mouse brain, the isolated brains were washed three times in PBS and transferred in 5 ml syringes filled with Fomblin (Solvay, Brussels, Belgium). A custom-made holder for the syringe was placed on a mouse holder (Medres, Cologne, Germany) and used in combination with a 9 cm resonator for transmission (Bruker, Ettlingen, Germany) and mouse quadrature surface coil (Bruker) for signal detection. All MR experiments were carried out on a Biospec 9.4 T animal scanner system with a 20 cm diameter bore magnet (Bruker) operated with ParaVision 5.1 software (Bruker). Transplanted cells were visualized performing a multi-slice multi-echo sequence using the following parameters: TR = 4,000 ms, TE = 12 ms, slice thickness = 0.7 mm, number of slices (coronal) = 10, FOV = 12×12 mm², matrix = 160×160, resolution 0.075×0.075 mm², bandwidth 50 kHz, echoes = 8. The acquisition time for these experiments was 10 min and 40 s. Quantitative T2 maps were calculated using a custom-made program developed in IDL (ITT Visual Information Solutions, Exelis Visual Information Soution, Boulder, CO, USA). The images obtained were analyzed with the ImageJ program (NIH, Bethesda, MD, USA).

Immunohistochemistry and Prussian Blue Staining

After MRI the brains were washed in PBS and transferred to 30% sucrose in PBS at 4°C until sunk. Coronal 20 μm-thick sections were serially cut with a cryostat, mounted on Superfrost Plus slides (Menzel Glaser, Fisher Scientific, Loughborough, England), and used for immunohistochemistry and Prussian blue staining.

For Prussian blue staining, the selected sections were stained with a 1:1 mixture of 10% K₄Fe(CN₎₆ (Sigma-Aldrich) and 20% HCl for 20 min. The sections were counterstained with 0.1% Nuclear Fast Red (Sigma-Aldrich) for 5 min, washed in PBS and distilled H₂O, mounted with HistoMount (Invitrogen) and coverslipped. After air drying, the brain sections were analyzed under bright field using an ECLIPSE E200 light microscope (Nikon Instruments).

For immunohistochemistry and immunocytochemistry, polyclonal antibodies against nestin (mouse monoclonal, diluted 1:200, Millipore, MAB353), MAP2 (chicken polyclonal, diluted 1:1,000, Abcam plc., Cambridge, UK, ab5392), GFAP (chicken polyclonal ab, diluted 1:250, Abcam, ab4674) and for oligodendrocytes O4 (monoclonal mouse anti-O4, dilution 1:50; Merck Millipore KGaA, Darmstadt, Germany, MAB345) were used. Briefly, brain sections/cells were incubated overnight at room temperature with the primary antibodies diluted in 0.2% Triton X-100 (Sigma-Aldrich) in PBS and 1% specific serum. The next day the sections/cells on glass slides were rinsed three times with PBS and then incubated for 2 h with the secondary antibodies diluted 1:500 in 0.2% Triton X-100 in PBS (goat anti-mouse Alexa Fluor 488 (Invitrogen), goat anti-mouse Alexa Fluor 546 (Invitrogen) and goat anti-chicken Alexa Fluor 546 (Invitrogen)). Secondary antibodies were rinsed with PBS three times and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI 250 ng/ml; Roche, Basel, Switzerland) was used as a nuclear counterstain. Finally, the brain sections/cells were rinsed in PBS, air dried, mounted with Dako Fluorescent Mounting and coverslipped before examination with the confocal microscope (Leica SP8 X FLIM, Germany).

Nanoparticle Biocompatibility In Vitro Tests

The labeled cells were tested by MTT assay, CalceinAM/PI cytotoxicity assay and neurosphere assay.

MTT (Sigma-Aldrich) was added to the cell culture medium at concentration 0.5 mg/ml and the cells incubated for 45 min at 37°C in 5% CO₂/95% O₂. The formed formazan crystals were dissolved in DMSO (Sigma-Aldrich), after which optical density was measured at 595 nm using a Microplate reader (680 XR, Bio-Rad Laboratories, Japan). MTT data were expressed as a percentage of the average values of the control cells according to the equation:

C e l l v i a b i l i t y ( % ) = ( A 595 s a m p l e − A 595 b l a n k ) / ( A 595 c o n t r o l − A 595 b l a n k ) * 100

For the CalceinAM/PI cytotoxicity assay the dissociated cells were incubated with 0.1 µM calcein AM (Invitrogen) and 5 ng/ml propidium iodide (Invitrogen). The percentage of calcein AM-positive NSC was analyzed using Attune acoustic focusing cytometer (Applied Biosystems) and calculated using FlowJo vX.0.7 software.

For neurosphere assay, cells were cultivated as free-floating aggregates in suspension for 24 h in triplicates; 0.02 or 0.2 mg/ml of D-mannose(γ-Fe₂O₃) nanoparticles were added to the medium for 48 h. After 2 days the average size of the neurospheres were measured in 10 visual fields per well in triplicates.

Statistical Analysis

For each experimental group, data were evaluated separately for a minimum of three independent experiments. For the nanoparticle uptake mechanism flow cytometry, the data were based on quadruplicate of each individual experiment. Data from the different experimental groups were statistically compared using one-way ANOVA analysis with Tukey’s test or Dunnet’s test as post-ANOVA analysis (p < 0.05) provided in the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). Grubbs’ test provided in the GraphPad Prism software (GraphPad Software, Inc.) was used to compare groups in flow cytometry experiments. Data were presented as mean values ± standard deviation (SD).

D-mannose(γ-Fe₂O₃) Nanoparticles Label NSCs More Efficiently than Uncoated(γ-Fe₂O₃) Nanoparticles

To verify whether NSC labeling can be improved with D-mannose(γ-Fe₂O₃) in comparison to Uncoated(γ-Fe₂O₃) nanoparticles, the cells were treated for 48 h and subsequently stained by Prussian Blue (Fig. 1). The presence of nanoparticles within the NSCs was indicated by a formation of blue precipitates as a result of the reduction of ferric to ferrous iron. The results clearly indicate the presence of both nanoparticle types within the NSCs, but more abundant when labeled by the same concentration of D-mannose(γ-Fe₂O₃).

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Figure 1.

Nanoparticle internalization verified by Prussian blue staining. Prussian blue staining of neural stem cells labeled for 48 h with ascending concentrations of D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles. The blue precipitate represents the nanoparticles. Nuclear Fast Red staining showed the position of the nuclei. Scale bar: 50 µm.

Flow cytometry measurements were performed to quantify the observed visual difference in NSC labeling. The changes of the laser beam SSC allowed measuring the changes in intracellular density, which correspond to nanoparticle internalization. The nanoparticle uptake was dose-dependent, and at concentrations of 0.2 mg/ml, D-mannose coating significantly improved nanoparticle internalization compared with their uncoated counterparts (Fig. 2).

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Figure 2.

D-mannose(γ-Fe₂O₃) nanoparticles label neural stem cells (NSCs) more efficiently than Uncoated(γ-Fe₂O₃) nanoparticles. (A) Quantitative analysis of the changes in intracellular density of NSCs labeled with ascending concentrations of D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles for 48 h, performed by Overtone cumulative histogram subtraction of flow cytometry histograms. (B) Flow cytometry histograms of D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles labeling efficiency of NSCs (Black line – control, three blue lines – nanoparticle concentration of 0.002 mg/ml, two green lines – nanoparticle concentration of 0.02 mg/ml, one red line – nanoparticle concentration of 0.2 mg/ml). The asterisk indicates a statistically significant (p < 0.05) difference between same nanoparticle concentrations.

Transmission electron microscopy was used to confirm the internalization of D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles in the NSCs. TEM micrographs clearly displayed and confirmed that both D-mannose-coated and uncoated nanoparticles were located intracellularly (Fig. 3). After 48 h incubation with D-mannose(γ-Fe₂O₃) nanoparticles, nanoparticle aggregates were localized in structures surrounded by a membrane, probably trafficking toward lysosomes (Fig. 3B). In contrast to D-mannose(γ-Fe₂O₃) nanoparticles, Uncoated(γ-Fe₂O₃) nanoparticles were not found inside membrane-bound vesicles; instead, they were found as aggregates dispersed in the cell cytosol (Fig. 3C´). The nanoparticles were loosely arranged in groups, and individual black dots of particles could still be observed. We did not detect any nanoparticles adhered on top of the cell membrane.

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Figure 3.

Transmission electron microscopy of neural stem cells (NSCs) nanoparticle internalization. Transmission electron micrographs of (A) unlabeled neural stem cells (NSCs), (B) labeled for 48 h with 0.02 mg/ml of D-mannose(γ-Fe₂O₃) nanoparticles or (C) Uncoated(γ-Fe₂O₃) nanoparticles. Inserts (B´, C´) show higher magnification of the nanoparticle aggregates inside the cell cytoplasm. Scale bar: 1 μm (A, B, C), 500 nm (B´, C´).

To clarify which endocytotic pathway was involved in NSC internalization of D-mannose(γ-Fe₂O₃) and Uncoated(γ-Fe₂O₃) nanoparticles, different inhibitors of endocytosis were applied prior to nanoparticle treatment and their effects evaluated by flow cytometry. The NSCs treated with an inhibitor of actin-dependent macropinocytosis, cytochalasin D, decreased labeling, being unable to internalize the nanoparticles. No changes in labeling were found when phenylarsine oxide, nocodazole, or filipin were applied (Fig. 4). This indicated that the internalization of both types of nanoparticles was via actin-dependent macropinocytosis.

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Figure 4.

Neural stem cells (NSCs) internalize D-mannose(γ-Fe₂O₃) and Uncoated(γ-Fe₂O₃) nanoparticles through macropinocytosis. Flow cytometry histograms of NSCs labeled with D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles show changes in side scattered (SSC) light of the laser beam after pre-treatment with different endocytosis inhibitors: cytochalasin D (red), phenylarsine oxide (black), nocodazole (pink), and filipin (yellow). Unlabeled cells as controls (black), and non-pre-treated labeled cells as positive controls (blue).

D-mannose(γ-Fe₂O₃)-Labeled NSCs can be Efficiently Detected by ex vivo MRI after Transplantation Into the Mouse Brain

Having established an optimized labeling with D-mannose(γ-Fe₂O₃)-coated nanoparticles (0.02 mg/ml for 48 h) NSCs were transplanted into the mouse striatum. MRI was performed ex vivo and unlabeled NSCs were used as a control. A pronounced hypointense region attributable to D-mannose(γ-Fe₂O₃)-labeled NSCs was observed in the striatum in T₂-weighted images (Fig. 5B). The MRI nanoparticle hypointense signal allowed the visualization and localization of transplanted NSCs labeled with D-mannose(γ-Fe₂O₃) nanoparticles within the anatomically defined region of the transplanted tissue with a high spatial resolution. No MRI contrast signal was detected when unlabeled cells were transplanted in the control animals.

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Figure 5.

D-mannose(γ-Fe₂O₃)-labeled neural stem cells (NSCs) were efficiently detected by ex vivo magnetic resonance imaging (MRI), Prussian blue staining and immunohistochemistry. Calculated T2 maps MR images of isolated adult mice brains at 72 h after a unilateral striatal transplantation of (A) unlabeled control neural stem cells and (B) D-mannose(γ-Fe₂O₃)-labeled (0.02 mg/ml) neural stem cells show the hypointense signal (blue) at the location of the graft (arrow). Prussian blue staining positive for D-mannose(γ-Fe₂O₃) nanoparticles (C) performed on serial rostrocaudal sections cut from the same brain scanned with MRI confirms the location of grafted labeled cells. Immunostaining against nestin (neural stem cell marker, green; D), DAPI (nuclear stain, blue; E) and merged (F) performed on same rostrocaudal sections cut from the same brains confirms the location of grafted cells. MRI scale bar 1 mm. IHC and Prussian Blue scale bars: 10 µm.

To verify if the MRI hypointense signal can be attributed to the D-mannose(γ-Fe₂O₃)-labeled NSCs, Prussian blue staining, and immunofluorescence were subsequently performed on corresponding serial rostrocaudal sections cut from the same brains. The formed Prussian blue precipitates showed the presence of the iron nanoparticles distributed in the transplant region (Fig. 5C). Three days after transplantation, NSCs were still nestin positive, reflecting their immature phenotype (Fig. 5F).

D-mannose(γ-Fe₂O₃) Biocompatibility is Similar to Uncoated Nanoparticles

To compare the effects of D-mannose(γ-Fe₂O₃) versus Uncoated(γ-Fe₂O₃) nanoparticles on NSCs, the MTT tetrazolium and CalceinAM/PI assays were applied. The MTT assay showed viable cells with active respiratory mitochondrial activity (as mitochondrial succinic dehydrogenases reduce MTT into an insoluble purple formazan) ⁵⁷ . Both nanoparticle types decreased the number of active/viable NSCs in a dose-dependent manner (Fig. 6). The concentrations higher than 0.03 mg/ml gave significantly different results compared with untreated control cells. The decrease in cell viability was around 20% when the highest concentration of 0.2 mg/ml of nanoparticles was used.

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Figure 6.

Neural stem cell (NSC) metabolic activity/viability is similarly affected when labeled with D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles. NSCs were incubated for 48 h in ascending concentrations of D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles (n=12). Control NSCs were not treated. Cell metabolic activity/viability was determined by MTT tetrazolium assays immediately after incubation. *p < 0.05, when compared with control.

The CalceinAM/PI assay assessed the percentage of living cells (labeled with Calcein AM) and dead cells (labeled with PI). The mean number of living NSCs treated with D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles in all the tested concentrations were higher than 90% (Fig. 7). Both nanoparticles at the highest concentration tested (0.2 mg/ml) showed a significant decrease in NSC viability, but it was only less than 3%. Subsequently, although D-mannose-coated featured similarly in these tests to uncoated nanoparticles, due to the better labeling features, they were chosen for further biocompatibility testing.

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Figure 7.

Neural stem cell (NSC) viability is similarly affected when labeled with D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles. NSCs were incubated for 48 h in the absence (control) or presence of ascending concentrations of D-mannose(γ-Fe₂O₃) or Uncoated(γ-Fe₂O₃) nanoparticles. The cell viability was determined by flow cytometry (CalceinAM/PI cytotoxicity assay). *p < 0.05, when compared with control. There were no significant differences between same concentrations of D-mannose(γ-Fe₂O₃) and Uncoated(γ-Fe₂O₃) nanoparticles.

Neurosphere morphology was tested to verify if D-mannose coating influenced the NSC differentiation. The D-mannose(γ-Fe₂O₃) labeling slightly affected NSC potential to form neurospheres (Fig. 8). All spheres showed round or oval morphology, but their diameters were lower in D-mannose(γ-Fe₂O₃) treated culture when 0.2 mg/ml concentration was used (61.33± 1.08 μm vs. 57.12± 1.37 μm, p < 0.01).

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Figure 8.

D-mannose(γ-Fe₂O₃) labeling does not affect the neural stem cells (NSCs) potential to form neurospheres. Representative image of neurospheres formed from mouse NSC monolayers treated with D-mannose(γ-Fe₂O₃) nanoparticles for 48 h (B) or untreated controls (A). Bar chart showing the average sphere diameter per well. Scale bar 50 µm.

The differentiation potential of the NSCs after 0.2 mg/ml D-mannose(γ-Fe₂O₃) treatment was further analyzed by immunocytochemistry of the resulting cell lineages. The D-mannose(γ-Fe₂O₃)-labeled NSCs stained positive for nestin, showing no change in their neural progenitor fate in comparison to control unlabeled cells (Fig. 9). After culturing these cells for further 5 days they readily differentiated to astrocytes (GFAP+), oligodendrocytes (O4+), and neurons (MAP2+) in a similar way as untreated control cells (Fig. 10).

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Figure 9.

Labeling with D-mannose(γ-Fe₂O₃) nanoparticles does not alter neural stem cell (NSC) stemness. The neural progeny of NSCs labeled with 0.02 mg/ml D-mannose(γ-Fe₂O₃) nanoparticles was confirmed by immunostaining against nestin (green, NSC marker). Control NSCs were untreated. DAPI was used as a nuclear stain (blue). Scale bars: 10 µm.

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Figure 10.

Labeling with D-mannose(γ-Fe₂O₃) nanoparticles did not affect the multipotency of neural stem cells (NSCs). The presence of neurons (MAP2+), astrocytes (GFAP+), and oligodendroglial cells (O4+) in the differentiated cultures was assessed by immunofluorescence assay. Significant differences were not observed in the relative proportions of the different neural cell types or in their morphology when NSCs were treated with 0.02 mg/ml of D-mannose(γ-Fe₂O₃) nanoparticles with respect to untreated controls. Scale bars: 10 µm.