Magnetically Triggered Release of Entrapped Bioactive Proteins from Thermally Responsive Polymer-Coated Iron Oxide Nanoparticles for Stem-Cell Proliferation

Nanoparticles could conceal bioactive proteins during therapeutic delivery, avoiding side effects. Superparamagnetic iron oxide nanoparticles (SPIONs) coated with a temperature-sensitive polymer were tested for protein release. We show that coated SPIONs can entrap test proteins and release them in a temperature-controlled manner in a biological system. Magnetically heating SPIONs triggered protein release at bulk solution temperatures below the polymer transition. The entrapped growth factor Wnt3a was inactive until magnetically triggered release, upon which it could increase mesenchymal stem cell proliferation. Once the polymer transition will be chemically adjusted above body temperature, this system could be used for targeted cell stimulation in model animals and humans.


Nuclear magnetic resonance (NMR)
NMR was carried out in CDCl3 or DMSO-d6 on a JEOL ECS-400 spectrometer at 400 and 100 MHz for 1 H and 13 C NMR, respectively. A 400 MHz field strength was used with 8 scans across a scan range of -2 to 12 ppm and scan acquisition time of 2 s for 1 H NMR. A 100 MHz field strength was used with 256 scans across a scan range of -60 to 240 ppm and scan acquisition time of 1 s for 13 C NMR. Peak assignment was carried out using Structure-based Predictions in Nuclear Magnetic Resonance Spectroscopy (SPINUS) software 4 using default parameters online (http://joao.airesdesousa.com/spinus/) to simulate spectra for comparison to experimental data.

Transmission electron microscopy
Nanoparticle suspension in dH2O was sonicated for 15 min, one drop deposited onto 3 mm holey-carbon-coated copper grids, air-dried and imaged on a JEOL 2011 microscope operated at 200 kV. Images were extracted using Gatan Digital Micrograph software.

Vibrating sample magnetometry (VSM)
10 mg/mL nanoparticles in water in were measured in an ADE Model 10 (Microsense) vibrating sample magnetometer equipped with a CF-1200 cryostat (Oxford Instruments) between 5 and 40°C. Magnetisation curves at each temperature were obtained by sweeping the field from -10 kOe to +10 kOe.

Magnetic susceptibility measurements and calculations
Magnetic susceptibility measurements were recorded on a Guoy balance (Sherwood Scientific, MK1 Magnetic Susceptibility Balance) using 10 mg/mL nanoparticles immobilised in a 1.5% agarose gel. Homogeneity was ensured by bath sonication throughout the sample preparation process. Magnetic susceptibility was calculated using: where is mass susceptibility, l is the sample length in the tube (cm), m is sample mass (g), c is the balance constant, R is the sample balance reading and Ro is the empty balance reading.

Equations a) and b) Magnetic susceptibility equations: a)
Determination of mass S4 susceptibility (cgs) of samples from Guoy balance readings; b) Conversion from cgs into SI units (m 3 kg -1 ).

X-ray photoelectron spectroscopy (XPS)
XPS was conducted by drop casting nanoparticles (10 mg/mL) in aqueous solution onto silicon substrates. After solvent evaporation, dense layers of deposited nanoparticles were probed using a monochromated Al Kα source (1486.6 eV, Omicron XM 1000) with a power of 220 W. Photoelectrons were detected using an Omicron EA 125 hemispherical energy analyser with a 2 mm entrance aperture and the sample normal oriented at 22.5° to both the X-ray source and entrance optics of the analyser.

Specific absorption rate (SAR) calculation
SAR was calculated using the following equation: is the mass of iron in the sample (g), Csol is the specific heat capacity of the solvent (CH2O= 4.184 J K -1 g -1 ), sol is the mass of solvent (g) and dT(K) / dt (s) is the slope of temperature vs time.

Thermal gravimetric analysis
10 mg dry nanoparticle sample was pyrolysed at 25-600°C on a STA 625 (PL Thermal Sciences) under nitrogen at a ramp rate of 10°C/min.

Grafting density calculation
Polymer grafting density on the iron oxide nanoparticle surface was determined using an adapted calculation for TGA-derived grafting density (σTGA). 2 Briefly, the total number of polymer chains was calculated from the TGA-measured polymer mass loss between 200-600°C (50.3%), the mass of an individual particle derived from its radius (3.15 nm) and density (5.18 g/cm 3 for magnetite), and the average polymer mass per chain (Mn of 12.99 kDa/6.02x10 23 ). The number of polymers per particle was then divided by the surface area of an individual particle calculated using the radius.

Magnetic heating
Samples cooled with a circulating fan were subjected to a 0.67 T, 108 kHz alternating magnetic field generated across a 10 mm gap in a proprietary instrument ( Figure S7, S8). Water-cooled copper coils around a MnZn ferrite core generated the field.

Protein-loading of coated nanoparticles
1 mg PNIPAM-coated nanoparticles and 1 µg apotransferrin (Sigma) or 1 µg Wnt3a (R&D Systems) were dissolved in 100 µL buffer A (20 mM HEPES, 100 mM NaCl, pH 7.4), and incubated at 37°C for 1 h while shaking, followed by 1 h at room temperature under shaking. Excess protein was removed by magnetic separation between four 1 h washes with buffer A containing 10 mg/mL RNase B (for apotransferrin) or with DMEM containing 10% FBS (for Wnt3a).

Protein release from coated SPIONs
Protein-loaded nanoparticles were incubated with 10 mg/mL RNase B in buffer A (for apotransferrin) or with 10% FBS containing DMEM (for Wnt3a) at the temperature and for the indicated times with or without magnetic treatment as described in the figure legends. Unless described differently in the figure legend, particles were magnetically separated at the time points before withdrawing 10 µL for analysis (for apotransferrin) or allowing cellular assays to complete (for Wnt3a). For magnetic heating assays, temperature was measured using an infrared thermocouple probe.

Protein analysis by SDS-PAGE and western blotting
Samples were separated on 10% SDS-PAGE gels prior to coomassie staining or western blotting. Coomassie staining was carried out according to the method of Fairbanks, 5 modified by boiling the gel in each of the staining/destaining buffers. For western blotting the proteins were transferred onto PVDF membranes by semi-dry transfer and probed with anti-apotransferrin (1:1000, Dako) in the presence of 5% milk in PBS-Tween20 buffer overnight at 4°C. Following secondary antibody binding, blots were imaged on a GeneGenius Chemi-imager (Syngene) after application of Immobilon HRP substrate (Millipore). Quantification was carried out using ImageJ software.
Cell culture Y201 hTert MSCs 6 and the MSC Wnt reporter line 7 were cultured in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) as published. Custom-made cell growth tubes for magnetic heating were designed in-house (35 mm x 20 mm x 8 mm, Figure S10), the base and lids of the tubes consist of polyether ether ketone (PEEK), and tube bases were secured to glass sides (flame polished) with a silicon O-ring.

Wnt reporter assays
Wnt reporter MSCs 7 were seeded at 17,500 cells/cm 2 and grown overnight before incubation with Wnt3a overnight at the indicated temperature. If necessary, SPIONs were removed by magnetic separation. Cells were washed three times with 200 µL PBS, then detached with trypsin and analysed by flow cytometry (CyAn ADP, Beckman Coulter). Results were gated for eGFP-positive (488 nm) live cells using the Summit software (v4.3.04, using default parameters).

MSC proliferation and viability
Y201 MSCs were seeded into custom-made tubes at 3500 cells/cm 2 . The following day SPIONs (or Wnt3a for calibration curve) were added, subjected to magnetic heating as indicated, then nanoparticles were magnetically removed, and the cells incubated at 37°C for 5 days to assay proliferation or 7 days to assess cell viability. Cells were then washed once with carbonate buffer (0.133 M Na2CO3, 0.066 M NaHCO3) before adding 150 μL 0.1% Triton-X100 in carbonate buffer. Cells were then freeze/thawed three times while applying manual agitation, and 50 μL lysate transferred into a black opaque 96 well plate (Greiner) and 50 μL picogreen reagent (Quant-iT PicoGreen dsDNA Reagent, Invitrogen, 50x diluted in 100 mM Tris, 1 mM EDTA, pH 7.5) was added. After rocking for 5 min at room temperature fluorescence was determined on a Clariostar plate reader (BMG Labtech) using 485 nm excitation and 538 nm emission wavelength.      The sample-specific area for the magnetic heating assays contained within the sealed box. Samples were positioned in an area that the alternating magnetic field was focused on. Annotations highlight specific components in this area, including: the thermocouple probe, which was inserted through an adapted inlet for temperature readings; and tubing, which was used to pump ice-cold water through coils surrounding the magnet. Figure S9. Release of the model protein apotransferrin from polymer coated SPIONs. a) 1.2 µg apotransferrin loaded into 1 mg coated SPIONs was incubated at 37°C in physiological buffer without the addition of any competing proteins, and at the indicated times the particles magnetically removed and the solution sampled. Samples were run on SDS-PAGE and coomassie stained. The apotransferrin in the first lane was run on the same gel, but not next to the release samples, hence the lane was moved, and this is indicated with a black divider line. The rest of the lanes contained wash samples, used to wash away excess apotransferrin following the loading step. b) Western blot analysis of the apotransferrin collected from the supernatant, following incubation of 1 mg apotransferrin loaded PNIPAM-coated nanoparticles in the presence of 10 mg/mL RNase B at pH 7.5 with or without magnetic heating (+/-MF). SPIONs were briefly collected on one side of the tube with a permanent magnet when the solution was sampled at the indicated time points. Temperatures measured during magnetic heating are shown in brackets above each time point. The sample without magnetic heating was maintained at 21°C. Densitometry of the apotransferrin immunoblot signal was used to quantify the release of apotransferrin. Error bars denote standard deviation, n=3. c) As in 'b' except that the magnetic field was turned off at the 10 min time point for the remainder of the experiment.  Emission at the wavelength of Texas-red was used to control for non-specific staining. b) Calibration curve derived from the scatter plots. Calibration curves were determined each time a Wnt-reporter experiment was performed. Each calibration curve was derived from data points determined using biological duplicates. c) Representative scatter plots of the Wnt-reporter experiments for which the quantification is shown in Figure 3b.  Figure S12. Temperature dependence of Wnt3a activity above 37°C Wnt3a was incubated for 10 min at the indicated temperature before incubating it with Wntreporter cells overnight. Wnt3a activity was then calculated using the eGFP-positive Wnt reporter MSC response relative to a control sample kept at 37°C. A line of best fit is shown. Figure S13. Dose response relationship between Wnt3a concentration and MSC proliferation. 1000 Y201 MSCs per well were cultured in the presence of the indicated Wnt3a concentration in a 96 well plate. The DNA quantities were then determined on the indicated days using picogreen staining and a standard curve derived from salmon sperm DNA. Error bars represent standard deviation, n=3, * indicates p<0.05 using Kruskal-Wallis One Way ANOVA on Ranks, followed by Dunn's post hoc test.