Engineered Biocompatible Nanoparticles for in Vivo Imaging Applications

Iron−platinum alloy nanoparticles (FePt NPs) are extremely promising candidates for the next generation of contrast agents for magnetic resonance (MR) diagnostic imaging and MR-guided interventions, including hyperthermic ablation of solid cancers. FePt has high Curie temperature, saturation magnetic moment, magneto-crystalline anisotropy, and chemical stability. We describe the synthesis and characterization of a family of biocompatible FePt NPs suitable for biomedical applications, showing and discussing that FePt NPs can exhibit low cytotoxicity. The importance of engineering the interface of strongly magnetic NPs using a coating allowing free aqueous permeation is demonstrated to be an essential parameter in the design of new generations of diagnostic and therapeutic MRI contrast agents. We report effective cell internalization of FePt NPs and demonstrate that they can be used for cellular imaging and in vivo MRI applications. This opens the way for several future applications of FePt NPs, including regenerative medicine and stem cell therapy in addition to enhanced MR diagnostic imaging.


fcc-FePt-silica-A:
The fcc-FePt-silica-A nPs were prepared by hydrolysis of tetraethylorthosilicate (TEOS) and the silica shell surface was further functionalized with (3-aminopropyl) triethoxysilane. 4 Reverse microemulsions were prepared by mixing under vigorous stirring 10 mL cyclohexane, 1.3 mL NP-5 and 50 µL DI H 2 O. 2 mg (~ 3-4 nmol) of FePt nPs were then dispersed in 1 mL cyclohexane and added dropwise into the reverse microemulsion. After 15 min, 80 µL TEOS was added dropwise. After another 15 min, 150 µL NH 4 .H 2 O (28-30 %) was added dropwise. The solution was kept under constant stirring at for 72 h. To form amine functionalized FePt-silica nPs, 100 µL of APTES was added after 48 h and kept stirring for another 24 h. The nPs were precipitated by centrifugation after addition of 3 mL of ethanol and 2 mL of methanol. The nPs were redispersed in 5 mL of ethanol and precipitated by centrifugation after addition of 10 mL of hexane. This step was repeated up to 6 times to completely remove the surfactant. FePt-silica nPs were stable both in ethanol and DI water, while FePt-silica-A nPs were stable in deionized water.

SI-I.2. Experimental Techniques TEM:
Transmission electron microscopy (TEM) images were recorded using a Gatan CCD camera on a JEOL JEM-2011 electron microscope operating at 200 kV. The chemical composition of FePt nPs was examined with energy-dispersive Xray spectroscopy (EDX) using an Oxford Link system installed in the JEM-2011 microscope.
XRD: Wide-angle powder X-ray diffraction (XRD) data were collected on a Stoe STADI/P powder diffractometer operating in transmission mode and with a small angle position sensitive detector. Incident radiation was generated using a Fe Kα1 source (λ=1.936 Å). The strongest (111) peak was fitted with Lorentzianshaped peaks using STOEwinXpow and Kaleida-Graph softwares to precisely determine the diffraction peak positions. The crystalline grain size, D XRD , of the FePt nPs was calculated according to Scherrer's formula: 5 θ λ cos 9 .
where D XRD is the " average" dimension of the crystallites, λ is the wavelength of the X-ray source (for Fe source is equal to 0.193604 nm), B is the full width at half maximum of the peak intensity, θ is the glancing angle. The atomic composition of FePt NPs was calculated based on the linear relations between lattice constant, a, and Fe percentage, Fe %, reported by Bonakdarkpour et al. 6 From SI- Figure 1, the following equations can be obtained: x %Fe < 40 % a = -0.0014 x %Fe + 3.929 SI-eq. (1) x %Fe > 40 % a = -0.0041 x %Fe + 4.039 SI-eq. (2) SQUID: A 5.0 Tesla Superconducting Quantum Interference Device (SQUID) from Quantum Design (MPMS XL TM ) was used to characterize the nPs magnetic properties. The nPs were dispersed in polyvinylpyrrolidone matrix (V Polymer /V nPs = 20) to prevent interaction between the nPs while the resulting sample was loaded into a low background gelatin capsule. Zero-Field Cooled and Field Cooled (ZFC/FC) measurements were completed as follow: the sample was first cooled from room temperature to 2 K without any external field, next a small field 100 Oe was applied and the nPs magnetization was recorded as the temperature was increased up 275 K. The FC curve was obtained by cooling the sample back to 2 K under a 100 Oe magnetic field. The magnetization was then measured while the temperature was increased up to 275 K. Hysteresis measurements were completed at temperatures of 2 K and 300 K. The magnetization of the gelatine capsules and the PVP matrix was subsequently subtracted.

ICP-OES:
FePt nPs were dissolved by addition of hydrochloric acid (Trace SELECT ® , ≥ 37 %) and nitric acid (Trace SELECT ® , ≥ 69.0 %) mixed in 3:1 as volume ratio and left overnight at room temperature. After full dissolution of the nPs, the solution was further diluted with deionised water to prepare inductively coupled plasma-atomic emission spectrometry measurement (ICP-OES, Perkin Elmer Optima 5300 DV).
The conversion of the weight concentration of FePt metal core into Fe molar concentration was completed according to the following equation: where [Fe] (mM) is the molar concentration of iron, C FePt (µg/mL) is the FePt core concentration, M w-Fe the molecular weight of the iron atom, R Fe is the mass ratio of Fe in FePt metal core. The mass of the ligand was neglected. The Fe x Pt 1-x nPs composition was found to be x ~ 0.43 by XRD and ICP-OES. The mass ratio of Fe in the nP core (R Fe ~ 0.18) was calculated with the equation below: A similar approach as described by equation 4 was used for fcc-FePt-silica-A nPs: where [Fe] (mM) is the molar concentration of iron, C FePt-silica-A is the concentration of FePt-silica-A in µg/mL, R ' Fe is the mass ratio of Fe in FePt-silica-A nPs which is ~ 0.01 according to eq. 5, Fe Fe yR R = ′ (eq. 5) where y is the mass ratio of FePt in FePt-silica-A nPs, i.e.
[Fe x Pt 1x ] y [silica-A] 1-y , which was measured experimentally by ICP-OES and found to be ~ 0.052. As an illustration, according to eq. 4 and 5, the weight concentration of FePt-silica-A nPs at 100 µg/mL corresponds then to ~ 0.02 mM of Fe in molar concentration.  Medicine and Dentistry) and was grown in RPMI-1640 medium (GIBCO, Invitrogen). All media were supplemented with 10 % fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin. Cells were grown under standard cell culture conditions in 5 % CO 2 at 37 °C to reach confluence of 60-70 % before subjected to any further treatments.

SI-I.3. Cell culture
Cellular labelling/uptake of FePt nPs and TEM: Cell culture media were removed and replaced with the media containing FePt-A (30 µg/mL). For control experiments, media without FePt-A was used. After 12-16 h of culture, cells were washed and fixed with 4 % (w/v) paraformaldehyde/2.5 % (v/v) glutaraldehyde buffered in 0.2 mol/L PIPES as a standard buffer solution. After 30 min in fixative the cells were scraped off the flasks in1 mL of fixative, transferred to Eppendorf tubes and cetrifuged at 14000 g for 20 min. Pellets were washed in 0.2M PIPES and post fixed in 1 % aqueous osmium tetraoxide for 1 h, washed in water, and dehydrated in graded ethanol and propylene oxide before embedding in Durcupan resin (Sigma) and polymerisation at 60 °C for 24 h. 70 nm sections were cut on a Leica UCT ultramicrotome, mounted on Pioloform coated 100 mesh copper grids, stained with uranyl acetate and lead citrate before being examined in a Tecnai 12 electron microscope. Images were recorded in Digital Imaging Plates and scanned in a Ditabis Micron scanner (Pforzheim, Germany).

Cell viability study by MTS assay:
The MTS CellTitre 96 AQueous One Solution Cell Proliferation Assay (Promega, Southampton, UK) was used to determine cell viability in control and FePt nPs treated cells. This colorimetric assay is based on the conversion of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxy-phenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) compound to a colored, soluble formazan product in metabolically active cells. Cells were seeded at densities 1,250-10,000 cells/well in 96-well plates depending on the duration of incubation time and cultured overnight. Cell culture media were then removed and replaced with the media containing the FePt nPs at various concentrations with the iron content being determined by XRD and ICP-OES. For control experiments, media without FePt nPs was used. MTS assay was performed at specific time intervals by addition of CellTiter 96 ® AQueous One Solution reagent to each well, in triplicate, and further incubation of cells with the reagent for 3 h at 37 °C in 5 % CO 2 . The colour development, i.e. absorbance, was read at 490 nm using a platereader (E-Max; Molecular Devices, CA, USA) and absorbance values of both labelled and control cells were subtracted by that of blank wells. Cell viability in labelled cells was expressed as percentage of that from the corresponding control cells.

Prussian blue staining and light microscopy analysis:
To gain insight in intracellular iron ions distribution, cells grown on glass coverslips or cytospun on glass slides were washed with PBS to remove the excess of ferumoxides and then fixed with 4 % para-formaldehyde for 30 min. They were then washed with double distilled water (ddH 2 O) and incubated for 30 min with 2.5 % potassium ferrocyanide in 2.5 % HCl to produce Fe(III) ferrocyanide (Prussian blue). Samples were then counterstained with nuclear fast red for cell nuclei. Cells were examined using a Zeiss microscope Axiovert 200 (Zeiss, Oberkochen, Germany) at x20, x40 and x63 magnification and Axiovision 4.6 software (Zeiss). Cells were considered Prussian blue positive if intracytoplasmic blue granules could be identified.

Preparation of Agarose Gel Phantoms for MRI of FePt nPs and FePt nP-labeled Cells:
In the gel MRI study, 1 % (w/v) low melting point agarose was dissolved in deionized water at 60-70 °C. The desired amount of nPs, i.e. Fe concentration as determined by XRD and ICP-OES was then added to the solution to obtain the targeted concentrations (up to 0.25 mM) and mixed gently. For cellular MRI, FePt labelled melanoma A375M cells were washed three times with PBS and harvested, suspended in low melting point agarose gel (1 % in PBS at densities of 10 4 , 10 5 , 10 6 cell/mL, respectively. Samples were then transferred into 200 µL thin wall tubes. In both cases, samples were rapidly cooled on ice to form homogenous gel suspensions and kept at 4 °C until further experiments.

Quail Eggs Preparation:
Fertilized Japanese quail (Coturnix japonica) eggs were obtained from Rosedean, Huntingdon, Cambs. The eggs were then placed vertically with air sac uppermost in a humidified incubator with a temperature of 38 °C, this was referred to as Day 0. On Day 4, the shell above the air sac was pierced and cut away to remove the inner shell membrane and expose the embryo. The Day 4 embryos typically lay on their sides, the upper eye is visible allowing the solution containing the nPs to be injected into it using syringe with thin glass capillary needle. The top of the eggs were re-sealed with adhesive tape and then imaged. fcc-FePt-A nPs coated with either cysteamine or silica were added to cell culture media at concentrations ranging from 1-100 µg/mL. A control medium without nPs was also used.

SI-I.4. Micro-MRI
Micro-MRI data were acquired on a Bruker Avance FT NMR spectrometer with a wide bore 7.1 Tesla magnet resonating at 300.15 MHz for 1 H, fitted with Bruker micro-imaging magnetic field gradients. A birdcage radio frequency resonator with an internal diameter of 30 mm was used. MRI pulse sequences were taken from the Bruker Paravision® library. All acquisitions were made at 19 ºC. Two acquisition sequences were collected and averaged to improve the signal-to-noise ratio and reduce artefacts.
Relaxation measurements were determined from 128 x 128 axial planes across the samples with field of view of 30 mm, inplane resolution of 0.234 mm/pixel and image slice thickness of 1 mm. In a typical experiment, 9 tubes were studied simultaneously, gels with no contrast agent acted as a standard. A recycle time (T R ) of 12 s was used to avoid saturation. T 1 relaxation times were measured using an inversion recovery (180º-T I -90º) imaging pulse sequence; 8 different inversion times (T I ) that ranged from 100 to 15000 ms were applied and the echo time (T E ) was 4 ms.
T 2 relaxation times were measured using a CPMG (Carr Purcell Meiboom Gill) spin echo imaging pulse sequence; a train of 16 echoes was acquired and the delay (τ) between 180º pulses ranged from 8 to 15 ms depending on the samples. Single exponential relaxation times were calculated from experimental data. Each sample was studied three times and average Longitudinal and transverse relaxation rate calculated. r i is defined as longitudinal relaxivity (i=1) or transverse relaxivity (i=2), and calculated with the following equation: 9 where [CA] is the concentration of contrast agent, 1/T i (i=1 or 2) is the longitudinal or transverse relaxation rate with the presence of contrast agent, 1/T io is the relaxation rate of the medium in the absence of contrast agent.
The 128 x 128 x 128 three-dimensional RARE-8 (rapid acquisition with relaxation enhancement) experiments were acquired with T R of 250 ms and TE between 180º pulses of 25 ms. The field of view was 30 mm and in-plane resolution was 0.234 mm/pixel. The scanning time was 34 minutes.
3D representation of the embryo eye and major blood vessels were produced using Amira® software (Visage Imaging, Inc. San Diego, CA USA).

SI-II. FePt nanoParticles Characterization SI-II.1. Ligand exchange
Hexane and water are not miscible, and water which is denser (1.0 g/mL vs 0.7 g/mL) lies beneath the hexane phase. This provides a straightforward illustration of the effectiveness of the ligand exchange protocol described in the Section SI-I.1.
The oleic acid and oleylamine capped FePt nPs are only soluble in hexane giving a black color to the upper phase while the DI water remains colorless (SI- Figure 2A). In contrast, SI- Figure 2B presents the cysteamine capped FePt nPs which are soluble in deionized water dispersion leading the lower phase to be black whilst the upper hexane phase is remains transparent. After having replaced oleic acid and oleylamine with cysteamine, the nPs are soluble in water solution and stable for more than 8 months.
The efficiency of ligand exchange was then confirmed by Fourier transform infrared spectroscopy (FTIR spectra of nPs before and after ligand exchange are shown in SI- Figure 3, with all characteristic transmission peaks labelled and matching the literature values. 10 The upper spectra in SI- Figure 3 is characteristic of the nPs coated with oleic acid and oleylamine. The peaks at 1552 and 1713 cm -1 can be assigned to the bidendate (-COO-) and monodendate -CO-) modes of oleic acid binding. The shoulder at 1595 cm -1 is due to the scissoring mode of the molecularly bonded oleylamine. The two peaks at 2854 and 2924 cm -1 correspond to the symmetric and asymmetric -CH 2stretching modes of the oleyl group. The peak at 3005 cm -1 is characteristic of the stretching of =C-H. The original oleic acid and oleylamine coating of the FePt nPs surface was replaced by cysteamine through the ligand exchange protocol described in Section SI-I. 1. It efficiency is demonstrated by the bottom FTIR spectra in SI- Figure 3  absence of -COO-M and -CO-M, =C-H peaks along with ii) the dramatic decrease of the -CH 2 -stretching transmission mode. The shoulder at 2560 cm -1 is associated with -SH, 11 while the peaks at 3350 and 3448 cm -1 are consistent with the -NH 2 end group of the cysteamine. 12

SI-II.2. Silica Coating
The protocol associated with the silica coating was tuned to provide homogeneous SiO 2 shell as illustrated by the large scale TEM image in SI- Figure 4A. In addition, careful control of the experimental condition prevented the formation of silica nPs without any FePt as illustrated in SI- Figure 4B. The alteration of the magnetic properties of the nPs after the silica coating (SI- Table 1) is attributed to the formation of thin layers of iron oxide or iron silicide resulting in the reduction of the magnetic effective volume and/or an alteration of the material. In support of this interpretation, the strong base environment associated with the silica coating can partially oxidize the FePt surface resulting in a thin layer of softer magnetic material like iron oxide or iron silicide, which because of its thinness is not visible by XRD or TEM. [13][14] SI- Table 1. SQUID data of the FePt nPs. T b is blocking temperature, M s is the saturation moment and H c is the magnetic coercivity of the nPs.

Sample
T

SI-III. Cytotoxicity
SI- Figure 5A and B demonstrate that fcc-FePt-A nPs induce no cell viability damage at concentrations up to 30 µg/mL even after 7 days of incubation. Whilst, the same period of incubation with 60 µg/mL and 120 µg/mL nPs lead to a decrease of the cell viability of only about 80 % and 70 % cell viability, respectively.
The results obtain with MCF7 and U2OS cells are consistent with A375M cells data presented in the core of this publication. As expected the silica layer preserved the cell viability, with SI- Figure 5C showing that fcc-FePt-silica-A nPs did not decrease human cell proliferative activity under culture conditions at concentration up to 200 µg/mL.  Figure 6A illustrates macropinocytosis, one of the main non-specific endocytic pathways which involves the internalization of large areas of plasma membrane together with significant amounts of fluid. Large irregular vesicles called macropinosomes with size larger than 1 µm are generated when membrane protrusions fuse back with the plasma membrane as indicated by the arrows on SI- Figure 6A.

SI-IV. Cellular uptake SI-
SI- Figure 6B-C present TEM images of A375M cells incubated with fcc-FePt-A nPs at FePt metal core concentration of 30 µg/mL and fcc-FePt-silica-A nPs at nPs concentration of 50 µg/mL after 16 h. SI- Figure 6B shows macropinocytosis uptake mechanism. SI- Figure 6C shows intercellular presence of fcc-FePt-silica-A nPs. Both fcc-FePt-A and fcc-FePt-silica-A nPs demonstrated efficient non-phagocytic human cells internalisation by straightforward incubation, without any external allied force such as transfection agents or electroporation.

SI-V. MRI
As expected for high magnetic fields such as 7.1 T used in this work, 9 all samples (i.e. Feridex, fcc-FePt-A, fcc-FePt-silica-A) show weak effect on T 1 relaxation time of 1 H in 1 % agarose gel water solution (SI- Figure 7A). SI- Table 2 summarizes the relaxivity values r 1 and r 2 obtained in this work and compare them with iron oxide data previously SI- Figure 7.  published. The relaxivities values obtained with Feridex are consistent with literature's values. In addition and as presented in the core of the publication, it is noticeable that higher r 2 values were obtained with fcc-FePt based nPs compared with Feridex'. SI- Figure 7B shows that at a magnetic field of 5 T and a temperature of 300 K, fcc-FePt-A nPs have the highest magnetization of 112 emu/g of Fe; however, fcc-FePt-silica-A nPs and Feridex appear to have similar nPs magnetizations of about 80 emu/g of Fe. By design, the commercial SQUID magnetometer highest external magnetic field is 5 T, whilst MRI experiments were performed at 7.1 T. However, a higher nPs magnetization of fcc-FePt-silica-A nPs compared to Feridex can be expected by extrapolating the magnetization curves on SI- Figure 7 from 5 T to 7.1 T. It is indeed noticeable that Feridex has already saturated at 5 T, while the silica coated FePt nPs had yet to reach saturation magnetization. Figure 8 illustrates the effect of injecting embryo with fcc-FePt-A nPs (1 mg/mL, SI- Figure 8, top row) and fcc-FePt-silica-A nPs (27 µg/mL FePt core concentration, i.e. 560 µg/mL FePt-SI- Figure 8. Embryo injected in ovo with cell culture media (1 µL) containing 1 mg/mL fcc-FePt-A nPs (top row) and fcc-FePt-silica-A nPs at FePt core concentration 27 µg/mL (bottom row). 3D surface reconstruction of embryo eyes (blue) and blood vessels (red) showing the position in yellow of the transverse images (A); Dorsal view of transverse image of the embryo's head aligned through the eyes (B). 2D slices from 128x128x128 3D Rare-8 MRI data set of Day 4 quail embryo egg (T R ⁄ T E = 250 ⁄ 25 ms), field of view of 30 mm and pixel dimensions of 0.234mm/pixel. Label: y = yolk, b = brain, in-e = injected eye, un-e = uninjected eye; scale bar indicates 1mm. silica-A concentration, SI- Figure 8, bottom row). SI- Figure 8A are 3D surface reconstruction which have been processed to highlight embryo eyes in blue, the blood vessels in red, and the nPs in green. In both cases, the presence of the nPs solution produced a strong hypointensity in the images clearly visible because of the darker contrast when comparing with the eye which has not been injected (SI- Figure 8B).