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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Size-Tailored Biocompatible FePt Nanoparticles for Dual T1/T2 MRI Contrast Enhancement Ioana Slabu, Katharina Wiemer, Julia Steitz, Rebecca Liffmann, Benedikt Mues, Sabine Eisold, Tobias Caumanns, Joachim Mayer, Christiane K Kuhl, Thomas Schmitz-Rode, and Ulrich Simon Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00337 • Publication Date (Web): 15 Jul 2019 Downloaded from pubs.acs.org on July 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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Size-Tailored Biocompatible FePt Nanoparticles for Dual T1/T2 MRI Contrast Enhancement Ioana Slabua*‡, Katharina Wiemerb‡, Julia Steitzc, Rebecca Liffmannb, Benedikt Muesa, Sabine Eisoldb, Tobias Caumannsd, Joachim Mayerd, Christiane Kuhle, Thomas Schmitz-Rodea, and Ulrich Simonb*

aApplied

Medical Engineering, Helmholtz Institute, Medical Faculty, RWTH Aachen University, Germany. bInstitute

cInstitute

of Inorganic Chemistry, RWTH Aachen University, Germany.

for Laboratory Animal Science, Medical Faculty, RWTH Aachen University, Germany.

dCentral eDepartment

Facility for Electron Microscopy, RWTH Aachen University, Germany.

of Diagnostic and Interventional Radiology, Medical Faculty, RWTH Aachen University, Germany.

‡Equal

contribution

*Corresponding

authors

ACS Paragon Plus Environment

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KEYWORDS magnetic nanoparticles, MRI contrast agents, iron platinum, phase transfer, amphiphilic polymer coating

ABSTRACT

The development of new contrast agents (CAs) for magnetic resonance imaging (MRI) is of high interest especially due to the increased concerns of patient safety and quick clearance of clinically used gadolinium and iron oxide based CAs, respectively. Here, a two-step synthesis of superparamagnetic water soluble iron platinum (FePt) nanoparticles (NPs) with core sizes between 2 nm and 8 nm for use as CAs in MRI is reported. Firstly, wet-chemical organometallic NPs are synthesized by thermal decomposition in presence of stabilizing oleic acid and oleylamine. Secondly, the hydrophobic NPs are coated with an amphiphilic polymer and transferred into aqueous media. Their magnetization values and relaxation rates exceed those published for contrast agents already used for clinical application. Their saturation magnetization increases with the core size to approximately 82 Am2/kgFe. For 8 nm NPs the T2 relaxivity of approximately 221 (mM·s)-1 is 5 times larger than for the ferumoxides, and for 6 nm NPs the T1 relaxivity of approximately 12 (mM·s)-1 is slightly higher than that of ultra-small gadolinium oxide nanoparticles. The 6 nm FePt NPs are identified as excellent contrast agents for both T1 and T2 imaging. Most importantly, due to their coating significantly low cytotoxicity is achieved. FePt NPs prove to be a promising alternative to gadolinium and iron oxide nanoparticles showing high quality CAs characteristics for both T1- and T2-weighted images.


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INTRODUCTION Magnetic nanoparticles (NPs) are of great interest for a wide range of disciplines, such as magnetic fluids, catalysis, magnetic energy storage, spintronics and biomedicine.1-5 In the medical field, they are considered for use in tumor therapy as hyperthermia agents and as site specific drug delivery agents.6-9 In the field of diagnostic imaging, they are used as contrast agents (CAs) for magnetic resonance imaging (MRI).10-13 The efficiency of CAs is expressed as its relaxivity r1 or r2, which defines the ability to shorten the proton T1 or T2 relaxation times per millimole of the contrast agent. Commonly used CAs for T1-weighted imaging consist of gadolinium (Gd)-based complexes, whereas CAs for T2-weighted imaging are iron oxide NPs.14 Gd-based CAs cause a strong contrast enhancement. However, their short circulation time due to fast renal clearance, the toxic effects of Gd ions in patients with greatly impaired renal clearance, as well as the cerebral accumulation of de-chelated Gd explains the need for new CAs with improved patient safety.15-18 Iron oxide based CAs have not gained such broad acceptance in clinical practice, but are successfully used e. g. as liver-specific contrast agents to improve the contrast between normal liver tissue and hepatic deposits of cancer.19-20 Iron oxide NPs with sizes between 40 nm and 100 nm are cleared via the liver and, thus, can be applied to cell imaging of the reticulo-endothelial system (RES), e. g. macrophages in the liver or the spleen and, more importantly, to highlight intra-hepatic tissue that lacks these cellular components.21-22 Smaller iron oxide based CAs of approximately 20 nm have been proposed for use in MR lymphography.23 However, disadvantages of these CAs are the quick clearance through liver and spleen accumulation which are more pronounced for the CAs with bigger sizes.24

Recent investigations are directed towards the synthesis of new biocompatible contrast agents, which might be used for contrast enhancement in both, T1- and T2-weighted imaging. In order to


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avoid fast clearance, they must have small sizes, leading to longer intravascular half-life times. Such CAs must be able to generate sufficiently high magnetic gradient fields to cause the local field inhomogeneity required to induce not only T1 but also T2 contrast enhancement. In general, magnetic NPs are used as T2 CAs due to their high magnetic moment, strongly enhancing the r2 values. Kim et al. described the synthesis of 3 nm Fe3O4 NPs and identified them as good candidates for T1 CAs due to their small sizes and decreasing magnetic properties.25 Further, Wang et al. examined 1.7 nm and 2.2 nm Fe3O4 NPs which can be used as T1 and T2 CAs for MRI, respectively.26 They explain the suitability of such ultra-small NPs as T1 CAs with the high amount of unpaired electrons at the surface and with their reduced magnetic anisotropy. Recently, low molecular weight iron chelates were shown to be a promising alternative to Gdbased CAs for T1-weighted images.27 Current studies focus on the development of new T1 and T2 MRI CAs from new magnetic materials such as intermetallic phase FePt.28-31 Research was first focused on thin-films until a novel wet chemical route was presented by Sun et al. in 2000 making FePt NPs promising candidates for various applications in biotechnology and biomedicine.32 Their high crystallinity, very narrow size distribution, and especially their unique magnetic properties are favorable features to allow better contrast performance compared to iron oxide based CAs.33-36 However, the cytotoxicity remains a problem and mostly impedes their use in vivo.37-38 Biocompatible surfaces with functional sides for FePt NPs, which should diminish their cytotoxic effects, were realized by e. g. hydrophilic coating of the hydrophobic NPs or ligand exchange reactions. However, these procedures often resulted in aggregation of the hydrophilic FePt NPs or reduced magnetic response, but could not even fully suppress cytotoxic effects due to ion leaching.34-35, 3843

Minimizing cytotoxicity and tailoring the magnetic properties of FePt NPs is still required.


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In this paper, we report the transfer of hydrophobic FePt NPs with diameters of 2.2 nm, 3.2 nm, 6.2 nm, and 7.5 nm in aqueous solution by coating the NPs with an amphiphilic copolymer. The cytotoxicity of these NPs was tested on the HeLa cell line and contrast enhancement was evaluated with a 1.5 T clinical MR device for both T1 and T2 weighted measurements. The properties of the FePt NPs were compared to those of other FePt NPs reported in literature and iron oxide based CAs such as ferucarbotran (Resovist®) or ferumoxides (Ferridex®) which are clinically used.

EXPERIMENTAL SECTION Materials Platinum acetylacetonate (Pt(acac)2) (98 %, ABCR), iron pentacarbonyl (Fe(CO5)) (98 %, Aldrich) 1,2-hexadecanediol (Lancaster), dioctyl ether (99 %, Aldrich), oleylamine (70 % technical grade, Aldrich), oleic acid ( 99 %, Sigma-Aldrich), hexamethylendiamin (98 %, Aldrich) and 0.1 M sodium hydroxide (analytical grade, Merck) were used as received. In all cases ultrapure water (0.055 µS/cm) was used. Solvents (ethanol, hexane, chloroform) were degassed prior to use. If necessary, solvents were dried using standard laboratory techniques. For the coating process, the polymer poly(isobutylenmaleic) acid anhydride (85 % technical grade, Sigma Aldrich) was partially coupled with dodecyl amine (99.5 %, Fluka) according to literature.44 A brief description is also given in the supporting information. Cytotoxicity measurements were performed using low-glucose Dulbecco's modified Eagle Medium (DMEM) complete medium, containing 10 % heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 IU/mL penicillin, and


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100 mg/mL streptomycin as well as trypsin-EDTA, TritonTm X, PBS and 2,3,5-Triphenyl-2Htetrazolium chloride.

Syntheses of FePt NPs with different sizes The FePt NPs synthesis with different sizes relies on literature.33 Below, the synthesis of 3 nm FePt NPs is described as representative approach for the all synthesis in this work. The synthesis of 2 nm, 6 nm and 8 nm FePt NPs are depicted in detail in the Supporting Information. The 3 nm FePt NPs synthesis was carried out under inert argon atmosphere using standard airless conditions. A three-neck glass flask with magnetic stir bar and glass capillary for thermal probe was cleaned with peroxymonosulfuric acid before use. The three-neck flask was equipped under argon atmosphere with 197 mg Pt(acac)2 and 390 mg 1,2-hexadecanediol and was evacuated three times and flushed with argon. Afterwards, 20 mL of dioctylether were added into the flask and the solution was flushed with argon for 30 min. The mixture was heated up to 100 °C and 0.17 mL oleylamine, 0.16 mL oleic acid and 0.13 mL Fe(CO)5 were injected into the solution. Subsequently, the reaction solution was heated up to 295 °C and was kept under reflux conditions for 30 min. Afterwards, the solution was cooled to room temperature. 60 mL of degassed ethanol were injected in the reaction flask resulting in the precipitation of a black product. The resulting dispersion was centrifuged with 400 rpm for 10 min. The clear colorless supernatant was discarded and the black precipitate was redispersed in hexane. The precipitation step was repeated and the product was redispersed in a solution with 0.05 mL oleic acid, 0.05 mL oleylamine and 10 mL hexane. The solution was filtered (220 µm pore size) and stored under argon.


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Polymer coating of FePt NPs The transfer of the hydrophilic NPs into the aqueous phase, so-called polymer coating step, for all FePt NPs was performed according to literature protocols, which describe the calculation of the volume of polymer and crosslinker solution added to the hydrophobic FePt NPs.44-45 For the calculation, minor changes to the above-mentioned literature protocols were made as described below. The particle surface A (nm2) for a given particle amount was determined using the following equation: 𝐴 = 𝜋 ∙ 𝑛 ∙ 𝑁𝐴 ∙ 𝑑2𝑒𝑓𝑓,

(1)

with deff = dcore + 2dligand (nm) the effective diameter of one particle, n (mol) the amount of FePt NPs and NA = 6.02·1023 mol-1 the Avogadro constant. dligand denotes the thickness of the ligand molecule sand dcore the core size of the FePt NPs. By using equation 1, the volume of the polymer was calculated as follows: 𝑉𝑃 =

𝜋 ∙ 𝑛 ∙ 𝑑2𝑒𝑓𝑓 ∙ 𝐶𝑝

𝑅𝑃 𝐴

,

(2)

where Rp/A (monomer/nm2) is the number of polymer monomer per nanoparticle surface and Cp (mol/L) is the concentration of polymer monomer in the stock solution. For the interconnection of the polymer backbone around the particle a specific volume of crosslinker Vc was added during the polymer coating process which was derived from the following equation: 𝑉𝐶 =

𝜋 ∙ 𝑛 ∙ 𝑑2𝑒𝑓𝑓 ∙

𝑅𝑃 𝑟𝑐 𝐴 ∙ 𝑃

(3)

𝐶𝑐

where rc/P is the ratio between the number of crosslinker monomer to the total number of polymer monomer and Cc (mol/L) is the concentration of crosslinker monomer in the stock solution.


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The polymer coating step was carried out under standard airless argon conditions as follows: The FePt NPs were filled in a 25 mL Schlenk flask and the solvent was evaporated. The FePt NPs were dispersed in 5 mL chloroform, then a specific polymer volume was added (cf. equation 2). The reaction mixture was stirred for 30 min and then the solvent was evaporated. The corresponding volume of the crosslinker solution (cf. equation 3) was added and the resulting mixture was stirred for 30 min. Subsequently, the solvent was evaporated and, in this way, polymer coated FePt NPs were gained as a black solid. The polymer coated FePt NPs were then suspended overnight in sodium hydroxide (NaOH) solution (0.01 M NaOH , pH 12) and centrifuged for 7 min at 5 °C with 2000 rpm. The black supernatant was further purified via filtration through a centrifuge filter (100 kDa, Millipore) and washed 5 times with NaOH solution (pH 9) and ultrapure water. Infrared reflection absorption spectroscopy (IRRAS) measurements The measurements were performed for 7.5 nm FePt NPs dispersed (as shown before46) in hexane and polymer coated 7.5 nm FePt NPs dispersed in water using an A513/Q variable angle reflection accessory including an automatic rotational holder for the MIR polarizer. The setup and general procedure for the measurements were reported previously.47 The IR beam was polarized with a KRS-5 polarizer with 99 % degree of polarization. The angle of incidence was set to 801and p-polarized IR radiation was used to record the spectra. For the background measurements, a plasma cleaned platinum wafer was used and the sample chamber was purged with argon for 5 min. Pt wafers (2 x 1 cm2) were prepared by plasma sputtering technique onto a oriented Si wafer, depositing a 10 nm adhesion layer of Ti and 100 nm of Pt. Prior to functionalization or measurements, Pt wafers were cleaned in oxygen plasma ((p(O2) = 0.4 mbar, f = 40 kHz, P = 100 W, t = 2 min). For the sample measurements the NPs were immobilized on a


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Pt wafer overnight. During continuous purging 1024 scans were collected. The first scan was recorded at the moment when argon purging was started. The scans were averaged until the peaks arising from the water vapor in the sample chamber were compensated for which typically 800 to 1500 scans were necessary. The spectra were processed using the OPUS software (Bruker, Germany). Wherever necessary, scatter correction was applied to the spectra. Transmission electron microscopy (TEM) measurements High-angle annular dark-field scanning transmission electron microscopy (HAADF-TEM) was performed with a Zeiss Libra 200FE (Carl Zeiss, Oberkochen, Germany). The electron beam accelerating voltage was set at 200 kV. A droplet of the sample was trickled on a piece of Formvar and carbon coated copper grid and then air-dried under ambient conditions. The particle 1

1

diameter was determined using the cumulative log-normal distribution function CDF = 2 + 2 ⋅ erf

(

(ln (dc) ― µ 2σ2

) fit for N > 200 particles from each batch (size was determined manually with the

software Paint), where erf is the error function and dc the core diameter of the particles. With the σ2

parameters µ and σ obtained from fitting, the core diameter yields in dc = exp(µ + 2 ) with a standard deviation of SD = (exp (𝜎2) ― 1) ∙ (exp (2µ + 𝜎2)) .

Dynamic Light Scattering (DLS) and -potential measurements The measurements were performed at 20 °C using a Malvern Zetasizer NanoS (He-Ne laser with the wavelength of 633 nm) at a backscattering angle of 173°. Hydrodynamic diameter size distribution and zeta-potentials (-potentials) were calculated from diffusion coefficients using the software Malvern DTS v. 7.02. Fitting with the log-normal distribution function PDF = 𝟏

𝒆 𝒅𝐇𝝈 𝟐𝝅

―

(𝒍𝒏(𝒅𝐇) ― 𝝁)𝟐 𝟐𝝈𝟐

to the number-weighted size distribution of the hydrodynamic diameters


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yielded in the determination mean hydrodynamic diameter and its standard deviation as described above for the TEM measurements. In order to check their stability, particles were measured after they rested for 48 hours in PBS (phosphate-buffered saline). From these measurements, the mean hydrodynamic diameter and its standard deviation as well as polydispersitivity index (PdI) were determined. Atomic absorption spectroscopy (AAS) measurements For AAS, all samples were dissolved in aqua regia and the measurements were performed using an AA-6200 (Shimadzu). X-ray diffractometry (XRD) measurements XRD was applied to determine the crystal structure of the FePt NPs and their crystal size by measuring the diffraction patterns of X-ray photons at the atom lattices of the FePt NPs crystals. For that, an X-ray powder diffractometer STOE STADI P (STOE & Cie GmbH, Germany) with a Cu-K1-radiator ( = 1.54056 Å) was used. The XRD measurements for the FePt NPs resulted in an intensity profile of the diffracted X-rays. The Bragg angles were determined using the Pseudo-Voigt function as fitting model.48 The different Bragg angles were analyzed and compared to reference data from Powder Diffractometry Files of the International Center for Diffraction Data (ICDD) in order to identify the corresponding crystal structures for each FePt NPs sample (PDF # 00-029-0717). The average crystal diameter of the FePt NPs was determined from XRD measurements using the Scherrer equation.49

Cytotoxicity tests In vitro cytotoxicity tests (XTT-Assay) were conducted with HeLa cells. The HeLa cells were cultured in tissue culture flasks in low-glucose DMEM complete Medium. Confluent cells were


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harvested with the help of trypsin-EDTA. 2000 cells/well were seeded in 200 µL DMEM complete medium in a 96-well tissue culture plate and incubated for 48 h at 37 °C and 5 % CO2. After 48 h the medium was replaced with 100 µL of polymer coated FePt NPs samples diluted in DMEM to iron concentrations (cFe) of 9 mM, 33 mM, 83 mM and 34 mM. Control samples were measured consisting solely of DMEM complete medium (= 100 % survival) or 0.01 % Triton™ X (= 0 % survival). Cells were cultured for 48 h at 37 °C and 5 % CO2. Afterwards, the supernatant was removed; the cells were washed three times with 200 µL of sterile PBS/well and 80 µL/well fresh DMEM, 20 µL/well XTT solution was added and the cells were incubated for two to three hours until a color change was detected. The plates were measured at 450 nm with a reference wavelength of 690 nm. For the XTT analysis, cells incubated in cell media served as control and their measured optical density was taken as a reference value with 100% survival. Cells treated with 0.01 % Triton™ X served also as control giving a reference value for the optical density with 0 % survival. The IC50 value refers to the concentration of a FePt NPs sample that gives an optical density response half way between 100 % survival and 0 % survival. For the determination of IC50 values, the general equation for a sigmoidal dose-response curve, also commonly referred to as the “Hill equation” or the “four-parameter logistic equation” was used to fit the results. For this, the GraphPad Prism 8 software (GraphPad Inc., USA) was applied. Further, dialysis experiments performed to find out whether Pt ions are released which could be the reason for any toxic effects. For that, the hydrophilic FePt NPs were dialyzed against ultrapure water over 3 days. The subsequent analysis of the dialysis water was performed by AAS to assess the concentration of Fe or Pt ions.


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Magnetometry measurements The magnetization measurements were performed using a Magnetic Property Measuring System (MPMS) XL (Quantum Design, USA). For the determination of the susceptibility and saturation magnetization values, magnetization measurements were performed at 295 K varying the field strength from zero to 50 kOe. From the fit with the Langevin function (𝐿(𝜉) = coth 𝜉 ― 1

𝜉;

𝜉 =

𝜇𝐻

𝑘𝐵𝑇),

with 𝜇 = 𝑉𝑀𝑀𝑆 the particle magnetic moment, VM the mean magnetic volume, H the

applied magnetic field and kB = 1.38·10-23 J/K the Boltzmann constant), the saturation magnetization MS was determined. The zero field-cooled (ZFC) magnetization curves were obtained by measuring the magnetization in a magnetic field of 10 Oe (50 Oe for the smallest 2 nm particles) while the temperature was stepwise increased from 5 K to 295 K. The fieldcooled (FC) magnetization curves were obtained by magnetization measurements executed in the same field as for ZFC measurements while the temperature was stepwise decreased to 5 K. For the magnetization measurements performed at room temperature the NPs were measured dispersed in an aqueous solution and for ZFC and FC measurements, the NPs were diluted with 15 wt-% mannitol solution and freeze-dried. Magnetic resonance imaging (MRI) measurements The MRI measurements were conducted with a 1.5 T device from Philips (the Netherlands) using a 16-channel RF coil for excitation and signal reception. All samples were measured inside a polyacrylic acid gel phantom as described in ASTM standards.50 The T1 relaxation times were detected by using a Fast Field Echo (FFE) sequence (TR = 5000 ms, TE = 8.00 ms, TI delay times = [200, 400, 600, 800, 1000, 1200, 1400, 1800, 2000, 2200, 2500] ms, FOV = 340 mm x 340 mm, matrix dimensions 400 x 400, slice thickness 5 mm). Measurements of the T2 relaxation times were performed for the 2 nm to 6 nm particles with a Turbo Spin Echo (TSE) sequence


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(TR = 2000 ms, TE = [5.35, 10.70, 16.04, 21.39, 26.74, 32.09, 37.44, 42.78] ms, 8 echos, FOV = 340 mm x 340 mm, matrix 400 x 400, slice thickness 5 mm) and for the 8 nm particles with a Turbo Spin Echo (TSE) sequence (TR = 32 ms, TE = [3.06, 5.48, 7.89, 10.31, 12.73, 15.14, 17.56, 19.97, 22.39, 24.80] ms, 10 echos, FOV = 340 mm x 340 mm, matrix 400 x 400, slice thickness 1.5 mm).

RESULTS AND DISCUSSION Preparation of polymer coated hydrophilic FePt NPs Generally, ligand stabilized FePt NPs can be synthesized with different size and shape by adjusting synthesis parameters, e. g. the ratio between precursor and stabilizing agent.33, 51 Here, hydrophobic oleic acid and oleylamine stabilized FePt NPs with diameters of approximately 2 nm47, 3 nm31, 6 nm48 and 8 nm30 were synthesized based on literature reports and further functionalized by polymer coating.44-45 In Figure 1 the schematic illustration of the polymer coating process of hydrophobic FePt NPs is shown. This kind of phase transfer is advantageous compared to ligand exchange which was shown to lead to aggregate formation.35,

41

The

hydrophobic FePt NPs were mixed with a functionalized polymalic anhydride polymer. In CHCl3 solution, this procedure leads to a wrapping of the polymer chains around the hydrophobic NPs through hydrophobic interactions between the long alkyl chained stabilizing ligands (oleic acid and oleyl amine) and the brush like alkyl groups of the polymer chains.44-45 Subsequently, some retained anhydride groups were subject to an additional crosslinking with a diamine crosslinker, which resulted in interconnected polymer sequences around each NPs. After the association of the particles with the amphiphilic polymer and subsequent drying, the FePt NPs were hydrophilic and could be redispersed in aqueous solution.


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The hydrophilicity of the polymer coated FePt NPs results from remaining unreacted anhydride rings within the polymer’s backbone, which hydrolyzed during phase transition. At the given pH values, this lead to charged carboxylate groups at the FePt NPs surface, which electrostatically stabilize the NPs in solution. It should be noted that the coating can be used for a wide range of coupling reactions in organic solution before or after hydrolysis due to the carboxylic acid groups at the surface. Hence, the anhydride groups of the polymer at the NPs surface can be coupled to hydrophobic dyes and the carboxylic acid groups at the NPs surface in aqueous media can be functionalized e. g. via EDC/s-NHS coupling reactions with biomolecules.52

Figure 1. Schematic illustration of the polymer coating process of hydrophobic FePt NPs to hydrophilic FePt NPs and their phase transfer into aqueous solution.


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Basic characteristics of the FePt NPs The coated FePt NPs were analyzed by HAADF-TEM, XRD, DLS and AAS measurements. The results are listed in Table 1. The particles are negatively charged and show a narrow size distribution of core and hydrodynamic diameter as well as a nearly equal ratio between Fe and Pt. Table 1. Summary of the FePt properties: Fe to Pt ratio determined by AAS measurements, core diameter dc,TEM and dc,XRD determined by TEM and XRD measurements, respectively, hydrodynamic diameter dH and -potential determined by DLS measurements. σ denotes the standard deviation (cf. Supporting information for details, Figures S1, S2, S3 and Table S1). Ratio Fe/Pt

1:1.6

1:1.1

1:1.3

1:1.1

(𝑑c,TEM ± 𝜎𝑑c,TEM) / nm

2.2 ± 0.2

3.2 ± 0.3

6.2 ± 0.5

7.5 ± 1.1

(𝑑c,XRD ± 𝜎𝑑c,XRD) / nm

2.0 ± 0.1

2.4 ± 0.1

4.6 ± 0.5

7.5 ± 0.6

(𝑑H ± 𝜎𝑑H) / nm

7.2 ± 1.6

11.2 ± 2.4

12.2 ± 3.8

14.2 ± 2.7

( ± 𝜎) / (mV)

-38.0 ± 6.6

-63.6 ± 7.8

-67.1 ± 5.6

-45.5 ± 6.7

The HAADF-TEM images of the differently sized FePt NPs are shown in Figure 2 (the image quality in this figure was improved by using the software Image J 1.52n, Wayne Rasband, National Institute of Health, USA). These images reveal nearly monodisperse NPs with a mean core size (dc) between approximately 2 nm and 8 nm and a small standard deviation. The exact values are listed in Table 1. The hydrodynamic diameters (dH) of the polymer coated FePt NPs obtained by DLS measurements are between 7.2 nm and 14.2 nm. The values match the sizes expected from theoretical consideration of the core size plus the ligand size of approximately 1.7 nm and a polymer shell thickness of approximately 2 nm.44-45 The values of the


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hydrodynamic diameter and PdI values determined for particles dispersed in PBS are presented in Figure S3 and Table S1 in the Supporting Information. Except for the values of 2 nm FePt NPs, the hydrodynamic size values of all other FePt NPs dispersed in PBS are consistent with the values determined for FePt NPs dispersed in water, which demonstrates their stability. Further, the PdI value of 2 nm FePt NPs is greater than 0.7 indicating their polydispersed character probably due to the formation of small agglomerates. The -potentials are between -38 mV and 67 mV due to the carboxylic acid groups at their surface. The IRRAS measurements demonstrate the polymer bonding to the particle surface. Exemplary results are shown in the Supporting Information (Figure S9 and S10) for 7.5 nm FePt NPs. In the AAS measurements (cf. Table 1), the average composition FexPt1-x (x denotes the weight percentage of Fe in the FePt composition) of the 2 nm, 3 nm, 6 nm and 8 nm particles yielded Fe38Pt62, Fe48Pt52, Fe43Pt57 and Fe48Pt52, respectively. In Figure 3, exemplary XRD data is shown, also providing the determined Bragg angles for the FePt NPs with a core diameter of 7.5 nm. The XRD data for the other FePt NPs investigated here can be found in the Supporting Information (Figure S2). Peaks ranging from 38° to 42°, from 45° to 48°, from 65° to 70°and from 80° to 85° are observed, which is in agreement with the fcc FePt 〈111〉, 〈200〉, 〈220〉 and 〈311〉 reflections, respectively.53,54 The average crystal diameters determined from the XRD measurements are listed in Table 1 and are consistent with the corresponding crystal diameters determined by TEM measurements when taking into account the systematic uncertainties of the crystal size calculation from XRD measurements. The systematic uncertainties are caused by the use of the generalized form of the Scherrer equation and estimated to approximately 15%.55 For FePt NPs with a core diameter of 2.2 nm, no phase identification was possible due to the poor signal-to-noise ratio of the XRD data (see Supporting


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Information, Figure S2). This is attributed to their small crystal sizes. Nanomaterials typically show significant peak broadening and lower intensity, so that in many cases the very small differences in diffraction intensity cannot be detected by XRD measurements.51

Figure 2. TEM images of hydrophilic FePt NPs. (a) 2.2 nm FePt NPs, (b) 3.2 nm FePt NPs, (c) 6.2 nm FePt NPs, (d) 7.5 nm FePt NPs.

12000

(111)

11000

Intensity / counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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10000

(200)

9000 8000 (311) 7000

(220)

6000 5000

40

60

80

100

120

2 /°

Figure 3. XRD based determination of Bragg angle 2θ by fitting the peaks of the measured data to the Pseudo-Voigt function for dried for the 7.5 nm FePt NPs. The peaks correspond to the fcc FePt 〈111〉, 〈200〉, 〈220〉 and 〈311〉 reflections.


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In vitro cytotoxic evaluation The cytotoxic effects of the polymer coated FePt NPs were determined in HeLa cells using XTT-assays. The cells were incubated for 48 hours with one of 16 different solution types. These solution types were composed out of FePt NPs of the four different particle sizes, each with iron concentrations (cFe) of 9 mM, 33 mM, 83 mM or 34 mM. The results are displayed in Figure 4 and show the IC50 value of the hydrophilic FePt NPs for different sizes. The IC50 values of the self-synthesized polymer coated FePt NPs were compared with the IC50 values of hydrophilic FePt NPs found in literature, which were differently stabilized such as with polyethylene glycole ligands (PEG), with an iron oxide shell, and a L-DOPA coating, as well as with a solid porous CoS2 shell (adapted from literature data).36, 38, 52 The highest IC50 value of hydrophilic FePt NPs from literature can be observed for the silica coated FePt NPs with cFe = 35.8 µM (highest tested concentration, estimated and calculated from literature),52 which is below the one of the FePt NPs presented in this work. Their low cytotoxicity is probably based on widely suppressed leaching of toxic Pt ions due to the polymer coating, also preventing damage of the inorganic core. This assumption is supported by the AAS measurements results of the dialysis experiments performed against ultrapure water over 3 days with all types of FePt NPs: With the detection limit of 1 µg/L Fe or Pt, no Fe or Pt ions could be detected.


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7

10

6

10

5

10

4

IC50 / µM

10

3

10

2

10

1

10

0

10

-1

Fe PtCo S 2 (H eL (D Fe a, SP Pt24 E_ Cy h) a PE Fe s (H PtG( e SiO 20 la, 00 24 )) 2 -A h) b (A (A 27 37 80 5M ,2 ,M 4h c ) CF 7, U2 Fe Pt_ OS d 2.2 ) * nm Po Fe lym Pt_ 3.2 er nm Po Fe lym Pt_ 6.2 er nm Po Fe lym Pt_ 7.5 er nm Po lym er

10

Fe PtCO OH _9 nm

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Figure 4. IC50 values of the cytotoxic evaluation of polymer coated FePt NPs with different sizes compared to the ones known from literature tested on different cell lines. FePt@CoS2 (HeLa cell line, 24 h, cFe ≈ 0.13 µM), cysteine stabilized FePt NPs, 3.6 nm (HeLa, 24 h, cFe ≈ 13 µM), PEG stabilized FePt NPs, 9 nm (A2780 human ovarian carcinoma cell line, 24 h, cFe ≈ 22.4 µM), silica coated cysteamine stabilized FePt NPs, 3.6 nm FePt NPs, 17 nm silica shell (A375M melanoma cells, MCF-7 breast cancer cell line, U2OS bone osteosarcoma cell line, cFe ≈ 35.8 µM, *highest tested concentration). Adapted from literature.52


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Magnetic characterization In order to determine the magnetic properties of the hydrophilic FePt NPs field dependent magnetization measurements at room temperature (295 K) as well as ZFC and FC magnetization measurements were performed. All samples revealed a characteristic superparamagnetic behavior with vanishing coercitivity and remanence at room temperature. Figure 5 (a) shows an exemplary fit for the Langevin curve for the FePt NPs with a core diameter of 7.5 nm. The saturation magnetizations (Ms) values determined from the Langevin fit are listed in Table 2 (see Figure S4 of the Supporting Information, which shows all magnetization curves with Langevin fit). The saturation magnetization increases with the particle size up to approximately 82 Am2/kgFe. The dependence of the magnetic properties on the particle size is consistent with the findings from literature.56 In Figure 5 (b) exemplary ZFC and FC curves for the FePt NPs of 7.5 nm diameter are shown. From the peak in the ZFC curve the maximum temperature (TM) is determined. TM decreases from 60 K for the smallest polymer coated FePt NPs to 30 K for the biggest polymer coated FePt NPs (corresponding ZFC and FC curves are shown in Figure S5 of the Supporting Information). Assuming non-interacting particles, TM is used to obtain a rough estimation of the effective anisotropy energy EA  23.6 kBTM, which allows to calculate an effective anisotropy constant Keff = 6EA/(dc3), with dc as the mean core diameter derived from TEM measurement (cf. Table 1).57 This estimation gives the highest result for the 2.2 nm particles with a Keff value of approximately 100 kJ/m3 while for the 7.5 nm particles the lowest value of approximately 1 kJ/m3 was reached. The Keff for the 3.2 nm and 6.2 nm sample is summarized in Table 2. The effective anisotropy energy shows higher Keff values for smaller particles which is attributed to surface anisotropy effects and matches the findings in literature.5859

Even though, the Keff values deduced here are estimations, the trend of higher anisotropy


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energy for smaller particles is obvious. The high anisotropy in the order of 100 kJ/m3 found for the smallest particles corresponds to the highest values found in literature for the disordered phase of 4 nm Fe50Pt50 NPs stabilized with oleic acid and oleylamine.60 The anisotropy constant changes not only with size but also with stoichiometric composition.61 Here, the 7.5 nm, 6.2 nm and 3.2 nm FePt NPs have concentrations close to the stoichiometric Fe50Pt50 composition (Fe48Pt52 for 7.5 nm and 3.2 nm NPs; Fe43Pt57 for 6.2 nm NPs), while the 2.2 nm FePt NPs have a higher concentration of Pt (Fe38Pt62). Other than expected, only the Keff value of the 2.2 nm Fe38Pt62 NPs is in the same order of magnitude (100 kJ/m3) as the one of FePt NPs designed for biomedical applications reported in literature (6.9 nm Fe48Pt52, 3.3 nm Fe52Pt48, and 4.2 nm Fe70Pt30 NPs).62 The high Keff values described in literature were reached for higher iron concentrations in the stoichiometric composition than the one of the 2.2 nm Fe38Pt62 presented in this work. Nearly the same Keff values were also found for approximately 10 nm Fe80Pt20, Fe70Pt30 and Fe60Pt40 NPs which were used as MRI contrast agents.63 Table 2. Saturation magnetization Ms and effective anisotropy constant Keff values for the FePt NPs with different core diameters dc. σ denotes the standard deviation.

(𝑑c ± 𝜎𝑑c) / nm

(𝑀s ± 𝜎𝑀s) / (Am2/kg)

Keff (kJ/m³)

2.2 ± 0.2

52.8 ± 0.2

 100

3.2 ± 0.3

46.9 ± 0.2

 50

6.2 ± 0.5

69.9 ± 0.5

 10

7.5 ± 1.1

82.4 ± 0.6

1


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Figure 5. a) Magnetization with Langevin function fitted curves and b) ZFC/FC curves for hydrophilic FePt NPs with a core diameter of 7.5 nm. MRI measurements The performance of the synthesized polymer coated FePt NPs with different sizes as T1 and T2 CAs were investigated. From these measurements, we determined the longitudinal (r1) and transverse (r2) relaxivity values of the various samples by measuring the characteristic relaxation times of a concentration series and plotted the relaxation rates (R1 = 1/T1 and R2 = 1/T2) against cFe. Subsequently, we performed a linear fit and determined the slope defined as the relaxivity (r1 and r2) which represents the efficiency of the contrast agent. Figure 6 depicts the results for the samples with significant influence on either r1 or r2 values. The results for other samples are listed in Table 3 and the corresponding fits are included in Figure S6 in the Supporting Information. Exemplary MR images of the FePt NPs are displayed in the insets in Figure 6. The T1-weighted images appear brighter with increasing cFe demonstrating the strong influence on the longitudinal relaxation rate. While the bigger FePt NPs strongly influence the transverse relaxation which can be observed in the T2-weighted images getting darker with increasing cFe. For concentrations higher than 3 mM, complete signal loss was obtained. These measurements were not considered for the determination of the relaxivity values shown in Figure 6 and the ones


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listed in Table 3. For the particles with a diameter of 7.5 nm, no r1 value could be determined as a very quick signal loss occurred even at cFe of approximately 0.06 mM (cf. relaxation curves in Figure S7 in the Supporting Information) which is attributed to their high saturation magnetization values (see Table 2).

Table 3. Longitudinal r1 and transversal r2 relaxivity values of the polymer coated FePt NPs presented in this work as well as values of commercially used Fe3O4 and water-soluble ligand stabilized fcc FePt NPs acquired at different magnetic fields B0 of the MR scanner as reported in literature. Here σ war derived from the linear fit (see Figure 6).

Sample size)

(material;

particle (𝑟1 ± 𝜎𝑟1) / (s·mM)-1

(𝑟2 ± 𝜎𝑟2) / (s·mM)-1

B0 / T

Resovist® (Fe3O4; 25 nm)64

8.7 ± 0.5

61 ± 0.7

1.5

Resovist® (Fe3O4; 25 nm)64

2.8 ± 0.2

176 ± 0.9

4.7

Feridex ® (Fe3O4; 30 nm)64

4.7 ± 0.3

41.0 ± 4.1

1.5

Feridex® (Fe3O4; 30 nm)64

2.3 ± 0.1

105.0 ± 5.0

4.7

fcc-FePt-silica-A (3.6 nm)52

0.3 ± 0.1

210.0 ± 3.0

7.1

fcc-FePt-A (3.6 nm)52

2.5 ± 1.0

887.0 ± 32.0

7.1

fcc-FePt-TMAOH (9 nm)34

-

239

4.7

Fe38Pt62_2.2 nm

1.7 ± 0.1

8.2 ± 0.7

1.5

Fe48Pt52_3.2 nm

0.8 ± 0.1

19.6 ± 1.7

1.5

Fe43Pt57_6.2 nm

12.3 ± 1.0

113.0 ± 2.0

1.5

Fe48Pt52_7.5 nm

-

221.2 ± 5.5

1.5


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Figure 6. a) Longitudinal R1 and b) transversal R2 relaxation rates with linear fit plotted against increasing iron concentration of FePt NPs (R0 denotes the relaxation rate value of the reference sample without MNP). In the right top corners, details for low concentrations are shown. In the right bottom corners, exemplary a) T1- and b) T2-weighted MRI images with different Fe concentrations (increasing from left to right) for each of the examined particle sizes are shown.


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In Table 3 the relaxivity values of the self-synthesized samples are listed together with data of commercially used Fe3O4 NPs and other NPs reported in literature such as silica coated FePt NPs and other ligand stabilized FePt NPs. Remarkably, the r1 and r2 values of the FePt NPs presented in this work are as high or even higher than those from literature, although they were acquired with a clinical MRI device of 1.5 T and not 7 T as those from literature.49 It is known that the magnetic field strength has a great influence on the relaxivity and that according to the different field strength dependencies of r1 and r2, respectively, the r1/r2 ratios decrease with increasing magnetic field strength. These alterations of r1/r2 ratios are more pronounced for superparamagnetic particles.64 For ferucarbotran (Resovist®) and ferumoxides (Ferridex®) the r2 value increases by a factor of approximately 3 and 2.5, respectively, when measured with a 4.7 T instead of a 1.5 T MRI device (see Table 3). This indicates that the particles used in this work could perform even better as T2 CAs at higher magnetic fields, potentially exceeding the highest r2 values of 887 (s·mM)-1 previously reported in literature for a 7 T MRI device.52 Other articles on Fe58Pt42 (3.6 nm) Fe51Pt49 (6.1 nm) und Fe33Pt67 (12.8 nm) NPs investigated with a 1.5 T MRI device describe a dose dependent negative contrast: For the 12.8 nm FePt NPs, 86 % signal loss was achieved at an iron concentration of 1 mM, while for the 3.6 nm and 6.1 nm FePt NPs only a signal loss of approximately 30 % could be reached at 25 mM.36 The stronger signal loss for bigger particles at lower iron concentrations is in agreement with the observations in this work. However, the magnetization for FePt NPs depends on the ratio of Fe to Pt showing higher values for higher iron content. This could also influence the negative contrast in the above-mentioned examples. The results suggest that there is an optimum size of approximately 6 nm for the superparamagnetic core if the particles should act as both T1 and T2 contrast agents. This finding


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is in line with the data found in literature.65-66 The synthesized particles show good T1 contrast enhancing effects up to approximately 6 nm, especially the 6.2 nm particles have a very high r1 value even compared to gadolinium oxide NPs.67 For small particles, this can be explained by surface effects causing a paramagnetic behavior in a strong magnetic field.68-69 The paramagnetic behavior becomes obvious in the smooth increase of its magnetization curve compared to the saturation obtained for other FePt NPs investigated in this work (cf. Figure S5 in Supporting Information, which shows a comparison between all magnetization curves normalized to the saturation magnetization). Generally, the strong local field inhomogeneity caused by superparamagnetic NPs causes strong T2 relaxation time shortening which is much higher than the T1 relaxation time shortening. According to the Solomon-Bloembergen-Morgan theory, the T1 relaxation effects are related to the diffusion of water protons in the local field inhomogeneity of paramagnetic ions (outersphere theory) and to the temporary binding of water molecules to paramagnetic ions (innersphere theory).70 The main contribution to T1 relaxation arises in the inner-sphere from interactions between water protons and unpaired electrons. The most influential parameters for inner-sphere relaxations are the mean residence time (τM) of coordinated water, the molecular tumbling or rotational correlation time (τR) of the contrast agents, and the coordinating number (q) of water molecules. Ideally, short τM, long τR, and large q are expected for contrast agents to achieve high T1 relaxivity. For superparamagnetic NPs however, the inner-sphere contribution to the relaxation is minor compared to the dominant outer-sphere contribution. This outer-sphere relaxation is due to the dipolar interaction between the magnetic moment of the paramagnetic NPs and the proton spins. Such intermolecular mechanism is modulated by the translational correlation time of the magnetic core (τD) that takes into account the relative diffusion constant


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(D) of the magnetic center and the water molecule, as well as their distance of closest approach (d).70 For superparamagnetic NPs having a hydrophilic surface, an increase in r1 is often seen due to the presence of more long-lived water molecules in the outer-sphere. In addition, a large surface-to-volume ratio, which favors water accessibility is desired for a high r1.71 Due to this reason, ultrasmall NPs and NPs with reduced surface and shape anisotropy can be used as T1 CAs.72 It was previously reported that r1 decreases with increasing particle size of magnetic nanoparticles.73 This trend was explained by the higher magnetization values and the lower surface-to-volume ratio of bigger magnetic nanoparticles, which have less available metal ions with unpaired electrons. For these reasons, the T1 effect is enhanced for the small particles. However in this work, FePt NPs of 2 nm and 3 nm have lower r1 values compared to 6 nm FePt NPs. To explain this behavior a reformulated outer-sphere relaxation theory is needed which takes into account the high magnetic particle moments of superparamagnetic NPs under the influence of high magnetic fields. Due to the fact that the theoretical background of r1 for superparamagnetic NPs is adapted from the outer-sphere theory of paramagnetic ions, it always contains a low-field assumption (µB/kBT

T2

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