Coating and characterisation of magnetic nanoparticles
MNPs (Chemicell GmbH, Berlin, Germany) diluted with 9.5 mL deionised water [DI] were sonicated for 18 h. N-(3-dimethylaminopropyl)-N-ethylcarboimide hydrochloride (5.7 mg) and N-hydroxysuccinimide (11.4 mg) dissolved in 1.9 mL of 2-(N-Morpholino)ethanesulfonic acid hemisodium salt [MES] buffer (0.5 M) were added to the solution and stirred for 1 h at room temperature [RT]. MES buffer (0.1 M) was added, and the solution was centrifuged at 40,000 × g at 4°C for 0.5 h. MNPs were resuspended in the MES buffer (0.1 M, 19 mL) containing 0.95 mg PEI (molecular weight [MW] 750,000) and stirred at RT for 3 h. Glycine (25 mM) in phosphate-buffered saline [PBS] (9.5 mL) was added, and the solution was stirred for a further 1 h at RT. The nanoparticles were 'washed' with DI (three times), and the resultant MNP-PEI eluted from the solution using a high-powered magnet. The nanoparticle pellet was resuspended in 10 mL of DI.
MNP-PEI (4 mL) was added to 0.08 M sodium tetraborate (12 mL) followed by methoxypolyethylene glycol p-nitrophenyl carbonate (MW 5,000) (20 mg) with stirring for 3 h at RT in the absence of light. The resultant solution was washed with DI, and the MNP-PLL-PEGs eluted from the solution using a high-powered magnet. The nanoparticles were resuspended in 4 mL DI.
Nanoparticle concentration was determined using inductively coupled plasma [ICP] analysis and dispersed in DI before sonication for 10 min before subsequent measurements. Hydrodynamic diameters, polydispersity index and zeta potential measurements were carried out using a photon correlation spectrometer (Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, UK). All measurements were conducted in triplicate at 25°C, and an average value was determined. Prior to zeta potential analysis, standard control samples were run on the instrument.
Culture of cell lines
Two cell lines, SH-SY5Y (human neuroblastoma; ATCC, Manassas, VA, USA) and RAW 264.7 (mouse macrophage), were used in our study. Neuroblastoma cells are being used to represent cells present at the regenerative site during nerve regeneration, and macrophage cells are the body's first line of defence for the immune system, phagocytosing foreign bodies and cleaning the blood of unknown particles; hence, any particles administered to the site of nerve injury will encounter these cells. SH-SY5Y cells were cultured in 50:50 Dulbecco's minimum essential medium [DMEM]: Ham's F-12 media containing 10% heat inactivated foetal bovine serum [FBS], 2 mM L-glutamine and 1% penicillin streptomycin [Penstrep] (all purchased from Invitrogen Ltd., Renfrew, UK). RAW264.7 cells (kindly donated by Prof. Colin Watts and Dr Alan Prescott, College of Life Sciences, University of Dundee, Dundee, UK) were cultured in DMEM containing 10% FBS, 2 mM L-glutamine and 1% Penstrep. Cells were grown under standard conditions (37°C and 5% CO2) to reach a confluency of 70% to 80% before being subjected to any further experimentation.
Cellular uptake of nanoparticles measured by inductively coupled plasma
Cells seeded in 6-well plates and incubated with MNPs at final concentrations of 0, 1.56, 6.25 and 25 μg mL-1 (the concentration of MNPs used for all experiments indicates the concentration of Fe3+) for 24 h. The medium was removed, and the cells were thoroughly washed with PBS for three times, trypsinised and resuspended in medium. The cell number was counted using a haemocytometer, and cells were placed in eppendorf tubes (1 × 106 cells/tube). The cell suspensions were centrifuged at 800 rpm for 5 min, and the supernatant was discarded. Concentrated hydrochloric acid (100 μL) was added to the cells, and the tubes were incubated at 90°C for 0.5 h. The samples were cooled to room temperature and centrifuged at 1,500 rpm for 10 min. The supernatant was diluted with deionised water and run on an ICP instrument (Optima 7000V DV, PerkinElmer, Wokingham, UK). A calibration was carried out using iron standard solutions of 0.5 to 5 μg mL-1 (R = 0.9999). A control sample of deionised water was also run.
Observation of cellular uptake of nanoparticles by transmission electron microscopy
MNPs were added to cells cultured in 75-cm2 flasks (6.25 μg mL-1) for 24 h. The cells were washed with PBS (three times). A 10-mL fixative (4% paraformaldehdye, 2.5% gluteraldehyde in PIPES buffer, pH 7.2) was added to the flasks and incubated for 0.5 h at room temperature. The cells were scraped off the flask and centrifuged into a pellet. The pellet was set in resin, and micron-sized slices were cut. The specimens were viewed using transmission electron microscopy [TEM] (JEM.1200ex, JEOL Ltd., Herts, England, UK), and images were recorded in digital imaging plates and scanned in a Ditabis Micron scanner (Ditabis AG, Pforzheim, Germany).
Cell viability determination by MTS, CellTiter-Blue and CellTiter-Glo assays
MTS and CellTiter-Blue are colorimetric and fluorescent assays (respectively) used to measure cell viability via non-specific redox enzyme activity. CellTiter-Glo is a luminescent assay used to measure cell viability by ATP level. Cells (100 μL, 1 × 105 cells/ml) were seeded into a 96-well flat-bottomed plate (white for Titer-Glo) and incubated for 24 h at 37°C with 5% CO2. The medium was replaced with increasing MNP concentrations (1.56 to 25 μg mL-1) (in triplicates). The cells were incubated for 24, 72, 120 and 168 h (for 120- and 168-hr incubations, the medium was replaced at 72 h with fresh medium containing appropriate concentrations of MNPs). The cells were washed (with PBS, three times) and replaced with fresh medium (100 μL). MTS (20 μL) or CellTiter-Blue (20 μL) reagents were added to the wells, and the plate was incubated for 4 h protected from light. Absorbance (MTS) was recorded at 490 nm, and fluorescence intensity (CellTiter-Blue) was recorded (excitation 560 nm, emission 590 nm). To eliminate possible interference between MNPs and assay readings, cells treated with same concentrations of MNPs but without addition of assay reagents were used as blank wells. Both assays were measured on a Tecan M200 multimode plate reader (Tecan Austria GmbH, Grödig, Austria). CellTiter-Glo reagent was added to the wells (50 μL and 50-μL media) and incubated at room temperature for 10 min protected from light. The luminescence was recorded using the same multimode plate reader. As per MTS and Titer-Blue assays, blank wells (with no reagents) were measured for luminescence and deducted from the values in experimental wells. Values of viability of treated cells were expressed as a percentage of that from corresponding control cells. All experiments were repeated at least three times. All assay kits were purchased from Promega, Southampton, UK.
Trypan blue exclusion assay
Cells were seeded into a 12-well plate and incubated for 24 h at 37°C with 5% CO2. The cells were treated as previously described in MTS, Titer-Blue and Titer-Glo. The cells were washed with PBS three times and trypsinised. Trypan blue was added to a 100-μL cell suspension in an equal volume and incubated at room temperature for 5 min. The viable cells were counted using a Countess™ automated cell counter (Invitrogen, Ltd., Renfrew, UK). Values of viability of treated cells were expressed as a percentage of that from corresponding control cells. All experiments were repeated at least three times.
Reactive oxygen species assay
Cells were seeded into a 96-well plate (10,000/well) and incubated for 24 h. Cells were incubated with increasing MNP concentrations (1.56 to 25 μg mL-1) for 1, 4, 24 and 72 h. The cells were washed three times with PBS and incubated for 1 h with 100-μM carboxy-H2DCFDA (Invitrogen Ltd., Renfrew, UK) in PBS at 37°C protected from light. The cells were washed three times with PBS and incubated with a 100-μL serum-free medium for a further 0.5 h. The medium was replaced with PBS. The fluorescence intensity of the samples was measured at 560 nm (excitation) and 590 nm (emission) on a Tecan M200 microplate reader (Tecan Austria). The percentage of dichlorofluorescin [DCF] fluorescence was calculated in respect to control cells assumed to be 100%.a
Lipid peroxidation measurement by thiobarbituric acid reactive substance assay
Cells in an exponential growth phase were seeded into a 6-well plate and incubated for 24 h. The medium was replaced with increasing MNP concentrations (1.56 to 25 μg mL-1). After incubation, the medium was removed, and the wells were washed three times with PBS. Cells were trypsinised and resuspended in 0.5 mL PBS containing 0.05% butylated hydroxytoluene on ice. The cell suspensions were sonicated for 5 s three times at 40 V and were kept on ice. Malondialdehyde bis(dimethyl acetal) [MDA] standard solutions (0 to 5 μM) were prepared, and 100 μL of samples or standards was added to the Eppendorf tubes. Sodium dodecyl sulphate (100 μL, 2%) was added, and the tubes were incubated for 5 min at room temperature. Thiobarbituric acid (250 μL) was added to the eppendorf tubes before incubation at 95°C for 1 h. The samples were cooled on ice for 10 min and centrifuged at 3,000 rpm for 15 min at 4°C. The supernatant was pipetted into the wells of a 96-well plate, and fluorescent measurements were taken at 530 nm (excitation) and 550 nm (emission). The results were calculated as nanomoles of MDA per milligram of cellular protein. Protein content was determined by the addition of a 100-μL sample to a 3-mL Bradford reagent. The samples were mixed well at room temperature for 5 min, and absorbance was measured at 595 nm. The absorbance values were compared to a calibration curve carried out using bovine serum albumin, and the protein concentration was determined.a
Cell membrane integrity analysis
Cells were seeded into a 96-well plate (15,000/well) and grown for 24 h. The medium was replaced with increasing magnetic nanoparticle concentrations (1.56 to 25 μg mL-1). The plates were incubated for 1, 4, 24 and 48 h. After incubation 2 μL of lysis buffer was added to the positive control wells, and the plate was centrifuged at 1,500 rpm for 10 min at 37°C. After centrifugation, 50 μL of the supernatant was removed from each well and placed into a new plate, and 50 μL of a membrane integrity assay reagent was added to the wells. The plates were incubated for 10 min at 37°C protected from light. Twenty-five microlitres of stop reagent was then added to the wells, and the fluorescence of the samples was measured at 560 nm (excitation) and 590 nm (emission) on the microplate reader. The percentage of cytotoxicity with respect to the positive control wells was calculated, whereby the lysed cells were assumed to have 100% lactate dehyrogenase [LDH] release.a