Design and Characterization of Iron Oxide Nanoparticles ...

This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TMAG.2017.2707599, IEEE Transactions on Magnetics

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Functionalization of Iron Oxide Nanoparticles with HSA Protein for Thermal Therapy E. Mazario1, A. Forget1, H. Belkahla1,2, J.S. Lomas1, P. Decorse1, A. Chevillot-Biraud1, P. Verbeke3, C. Wilhelm4, S. Ammar1, J-M. El Hage Chahine1 and M. Hemadi1* 1

Interfaces, Traitements, Organisation et Dynamique des Systèmes, Université Paris Diderot, Sorbonne Paris Cité, CNRS-UMR 7086, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex 13, France 2 Nanomedicine Lab, EA 4662, Université de Bourgogne Franche-Comté, Besançon, France 3 UMR 1149 Inserm, Université Paris Diderot, Sorbonne Paris Cité, ERL- CNRS 8252, Faculté de Médecine, site Bichat, 16 rue Henri Huchard, 75018 Paris, France 4 Laboratoire Matières et Systèmes Complexes, Université Paris Diderot, Sorbonne Paris Cité, CNRS-UMR 7057, 10 rue Alice Domon et Léonie Duquet, 75205 Paris Cedex 13, France Over the past two decades important progress has been made in the development of nanomaterials especially for biomedical applications. The surface of magnetic nanoparticles (MNPs) can be modified and functionalized with a targeting agent in order to improve their longevity in the blood, their cell internalization and their efficiency. However, after this functionalization, some of their physical properties may be modified. In this study, spinel iron oxide single crystals 10 nm in diameter were elaborated and functionalized with HSA protein, to give a MNP-HSA nano-platform. Physicochemical studies were performed to evaluate this nanoplatform utility in nanomedicine. X-ray Photoelectron Spectroscopy (XPS) and Thermogravimetry (TG) were used to establish the presence of protein at the surface of MNP. MNP-HSA shows very good colloidal stability with a zeta-potential of -35.4 ± 0.6 mV at physiological pH (7.4). MNP-HSA is superparamagnetic and exhibits a saturation magnetization (Ms) of 63 emu.g-1 at 310 K and produces localized heat in an alternating magnetic field; the specific absorption rate (SAR) of an aqueous suspension of MNP-HSA is 123 W.g-1. To identify the relative contributions of Brownian and Néel relaxations in its heating process, MNP-HSA was immobilized in an agarose gel, where the SAR was found to be the same as in water. This indicates that Néel relaxation is the dominant nanoheating mechanism. Functionalization, Magnetic hyperthermia, Magnetic nanoparticles, Targeting protein.

I. INTRODUCTION

M

ultifunctional nanomaterials are increasingly finding applications in nanomedicine. In particular, magnetic nanoparticles (MNPs) are extensively applied in biomedical research as one of the most challenging strategies for cancer treatment. Indeed, they have been used in theranostics for their simultaneous therapeutic and diagnostic capabilities: for hyperthermia [1], for magnetic resonance imaging (MRI) [2], and also for drug delivery [3]. Iron oxide MNPs have attracted much attention, especially those with diameters less than 20 nm, which are superparamagnetic [4, 5]. They can be manipulated by external magnetic fields, which makes them suitable for magnetically assisted biomedical applications. Some of the more interesting features are their biocompatibility, low toxicity, stability and biodegradability [6, 7]. MNPs need to be functionalized in order to give them high colloidal stability and to facilitate their internalization. Furthermore, functionalization with targeting agents such as proteins, DNAs, antibodies, etc. may further enhance their suitability for biomedical applications [8]. Among the proteins that have been extensively used for drug targeting and drug delivery, there is Human Serum Albumin (HSA), the most abundant protein in human blood [9]. HSA has been used as a drug carrier in the field of chemotherapy to improve the passive tumor targeting properties of anti-cancer drugs [10] and also in the field of diabetes to target the insulin receptor Corresponding author: Miryana Hémadi (e-mail: [email protected]).

[11]. This protein constitutes therefore an attractive targeting agent. In this work, attention is given to the design of an effective nanomaterial by combining the potential of MNPs for hyperthermia and the biological characteristics of HSA for efficient and safe treatment. We describe the synthesis and characterization of a nano-platform based on spinel iron oxide single crystals coupled to HSA. In practice, the nanocrystals were functionalized first by 3-Aminopropyl TriEthoxy Silane (APTES) in order to obtain amino functions at their surface (MNP-APTES). HSA with its carboxylic groups was then grafted via amide bonds onto MNP-APTES. The resulting nano-platform, MNP-HSA, as well as the raw MNPs and MNP-APTES, were characterized by several techniques: Xray Photoelectron Spectrometry (XPS), Thermogravimetry (TG) and zeta-potential measurements. In order to show a suitable efficiency in the intended applications, MNPs should have the greatest possible saturation magnetization (Ms); routine magnetometry was carried out on raw MNPs and MNP-HSA. Finally, the suitability of MNP-HSA for thermal application was checked by magnetocalorimetric measurments, focusing on their specific absorption rate (SAR), which usually defines ability of a magnetic matter to convert the magnetic energy into heat in an alternating magnetic field (AMF), is defined by the specific absorption rate (SAR)[12]. SAR was calculated under different conditions: in water at different frequencies and magnetic magnitude, and in agarose gel. This last experiment was designed to determine the relaxation process by which heat is generated within the magnetic core.

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𝐶𝑤𝑎𝑡𝑒𝑟 𝑉𝑠𝑎𝑚𝑝𝑙𝑒 𝑚𝐹𝑒

∙

-1

𝑑𝑇 𝑑𝑡

, where Cwater is the specific capacity

-1

of water: 4185 J.L .K , Vsample is the sample volume in L, and mFe is the total mass of iron in the sample in g.

III. RESULTS AND DISCUSSION HSA protein grafting was characterized first by XPS. Fig. 1 shows the survey spectra of the MNPs and MNP-HSA: the nitrogen peak (399.9 eV) increases from the MNPs, where the nitrogen/iron ratio is nil, to 0.48 for MNP-HSA (insert of Fig. 1A). Moreover, the intensity of the iron peak (710.6 eV) decreases in MNP-HSA. Since the XPS analysis depth does not exceed a few nanometers this clearly demonstrates the presence of protein at the surface of the MNPs. FTIR spectra (Fig. 1B) of MNP-HSA (red) show clearly also the presence of protein at the surface of MNP; the absorption bands at 1642 cm−1 and 1538 cm−1 are related to the amide I (C=O

A

Nitrogen

Counts (a.u)

XPS was performed with an ESCALAB 250, Thermo VG Scientific spectrometer equipped with a monochromatic Al K α X-ray source. Samples were stuck onto sample holders using conductive double-sided adhesive tape and pumped overnight in the fast entry lock at ~10−10 mbar prior to transfer to the analysis chamber. FTIR transmission spectra were acquired on a Nicolet Magna-IR860 spectrophotometer on MNP and MNP-HSA samples dried and incorporated into KBr pellets. .TG was performed in a Setaram TGA92 system from room temperature up to 1000 °C in air at a heating rate of 20 °C min-1. The zeta-potential or electrokinetic potential (ξ) was measured as a function of pH with Malvern Nano Zetasizer equipment. The concentration of all samples was fixed at 0.1 g.L-1. Magnetic studies on raw MNPs and MNP-HSA were conducted with a Quantum Design MPMS-5S SQUID magnetometer. The magnetization was recorded at 310 K as a function of the magnetic field (H) from –50 to 50 kOe. The iron concentration in MNP-HSA was determined by Prussian blue assay: MNP-HSA was first degraded in acidic medium (HCl, 10 M) for one hour. This degradation leads to the production of iron(III) complexed with Cl− ions from HCl. These Fe(III) in the presence of K4FeII(CN)6 (yellow) form a Prussian blue complex, KFeIII[FeII(CN)6], which absorbs at 720 nm with a molar absorption coefficient of 8600 L.mol−1.cm−1. From this absorption the iron concentration was determined. Dispersions of MNP-HSA in water with iron concentrations of 14 mM and 28 mM were used for magnetic hyperthermia (MH) measurements. MNPs were also fixed in 2% agarose gel by heating an agarose suspension to its boiling point, then mixing the particles with the suspension and allowing the solution to cool and solidify in a plastic Eppendorf tube. MH was monitored on a nanoScale Biomagnetics in magnetic

SAR =

Counts (a.u)

10 nm-sized MNPs were prepared by the polyol method [13]. Typically, an appropriate amount of iron(II) acetate as metal precursor and triethyleneglycol (TEG) as solvent [14]. With mechanical stirring the reaction mixture was heated at a rate of 6 °C min-1 from room temperature to the boiling point (230 °C) and maintained under reflux for 1 h. A black magnetic powder was recovered by centrifugation after cooling to room temperature. MNPs were then functionalized with APTES according to a well-established protocol [15]. They were washed several times with ethanol and dried overnight at 60 °C. To a solution of HSA (50 µM) in 2-(Nmorpholino)ethane sulfonic acid (MES 100 mM, pH 6) were added microvolumes of N-hydroxysuccinimide (NHS, final concentration: 3.75 mM) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, final concentration: 1.5 mM) in order to activate the carboxylic acid groups and obtain a reactive HSA NHS ester. MNPs functionalized with APTES were then added to the mixture of protein, and an amide bond was formed between the reactive ester of the protein and the amino groups of MNP-APTES in a buffer at pH 8, yielding the MNP-HSA nanohybrid.

fields up to 180 G at frequencies in the range of 144-471 kHz and at a frequency of 471 kHz in the range 90-180 G. For all the measurements, the temperature elevation was recorded in real time (every second) with an infrared thermal imaging camera (FLIR SC7000). The temperature elevation was measured as a function of time (dT/dt) for the first 30 s after application of the AMF in order to evaluate the heating effect in terms of SAR, which is defined as the power dissipation per unit mass of iron (W.g-1) and is expressed as eq. 1:

393 396 399 402 405 408

Binding energy (eV)

Fe3O4

N

Fe3O4 - HSA 1000 110

B

800

600

400

200

0

Binding energy (eV)

100

Transmittance (%)

II. MATERIALS AND METHODS

90

1640 cm

80

1642 cm

70 60 40 20

-1

1538 cm

-1

Iron oxide skeleton

50 30

-1

3420 cm

-1

MNP MNP - HSA

3500 3000 2500 2000 1500 1000 -1

500

Wavenumber (cm ) Fig. 1. A- XPS survey spectra for Fe3O4 nanoparticles (MNPs) in black, and MNPs functionalized by HSA (MNP-HSA) in red. B- FTIR spectra of KBr pellets of MNP (black) and MNP-HSA (red).

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70 60 50

310 K

40

60 -1

30 20

MNP MNP-HSA

10 0

Zeta Potential (mV)

60

0

MNP MNP - APTES MNP - HSA

40

M (emu g )

The grafting of HSA onto MNPs was also characterized by TG, where the organic weight loss was 9.6%. By subtracting the weight loss relative to APTES and to HSA, and taking into account the average diameter of the MNPs (10 nm), we estimate that 4 molecules of HSA protein were grafted at the surface of one MNP. Fig. 2 shows the dramatic evolution of the zeta-potential of MNPs, MNP-APTES and MNP-HSA with pH. At physiological pH (7.4), ζ varies from (-31.5 ± 0.5) for MNPs to (+4.3 ± 0.3) for MNP-APTES, and to (-35.4 ± 0.6) for

this approach, the conversion of magnetic energy into heat in an alternating magnetic field (AMF) is mainly due to thermal fluctuations within a nanoparticle. Heat can be generated by two different processes: relaxation and hysteresis loss. The magnetic moment is relaxed either through Néel or Brownian mechanisms [19]; both are described by characteristic

M (emu.g-1)

stretching) and the amide II (CN stretching and NH bending), respectively, of the HSA secondary structure. The absorption bands in MNP (in black, Fig. 1B ) and MNP-HSA (in red, Fig. 1B) at 634 and 579 cm−1 are typical of Fe–O in iron oxide nanoparticles[16]. The bands at 1640 and 3420 cm−1 in MNP (black) are those of water (δOH and νOH).

40

MNP-HSA

20 0 -20 -40 -60

-1200 -600

0

600 1200

Field (Oe)

10000 20000 30000 40000 50000 60000

Field (Oe)

20

Fig. 3. First magnetization recorded at 310 K on raw MNPs with Ms = 69.3 emu.g-1 and on MNP-HSA with Ms = 63.0 emu.g-1.

0

relaxation times [20]. In Néel relaxation, the direction of the magnetic moment rotates rapidly in a given direction relative to the crystal lattice under the constraint of the magnetic anisotropy energy. This type of relaxation occurs when the measurement temperature is above the blocking temperature and the measurement time is longer than the relaxation time. The Néel relaxation time (τN) is given by, eq. 2: 𝐾𝑉 𝜏𝑁 = 𝜏0 𝑒𝑥𝑝 ( 𝑚⁄𝑘 𝑇 ). 𝐵 It depends exponentially on the product of the magnetic anisotropy energy density (K) and the magnetic core volume (Vm), where kB is the Boltzmann constant, T is the temperature, and τ0 is 10-9 s [21]. Brownian relaxation is due to the physical rotation of particles in a medium. This relaxation is hindered by the viscosity of the medium that tends to reduce the movement of the particles. With this relaxation, the magnetic moment rotates when the measurement temperature is less than the blocking temperature and the measurement time is shorter than the relaxation time [19]. Brownian relaxation (τB) is given by, eq. 3:

-20 -40 -60

3

4

5

6

7

pH

8

9

10

11

Fig. 2. Variation of the  of MNPs (black), MNP-APTES (red), and MNPHSA (blue) with pH.

MNP-HSA. This variation indicates that each step in the design of functionalized MNP was a success: raw MNPs have high colloidal stability at pH 7.4. When APTES is added there is an increase in ζ (less negative) explained by the presence of amino functions in the APTES molecule (pKa = 7.5) [17]. The grafting of HSA at the surface of MNP-APTES modifies the charge of the surface, which is expressed as a decrease in ζ due to the lower isoelectric point of the protein (pI = 4.8). The zeta-potential of MNP-HSA indicates that the nano-platform has high colloidal stability which will ensure its transport in the blood. Both these characterizations demonstrate that HSA is well grafted onto the MNPs. The magnetic properties of the nano-platform were investigated at 310 K, the body temperature, and compared to that of raw MNPs. The insert in Fig. 3 confirms that MNPHSA is superparamagnetic, with an Ms of 63.0 emu.g-1. The presence of protein at the surface of the MNPs is revealed by a 9.1% decrease in the Ms because of the diamagnetic contribution of HSA. This is consistent with the results obtained by TGA. By magnetic hyperthermia, the temperature of the local environment of a tumor is raised, resulting in a change in the physiology of diseased cells and finally leading to apoptosis [18]. The MNP-HSA nano-platform was tested as a nanoheater with a view to potential therapeutic applications. In

τB =

3ηVh kBT

.

This is proportional to the fluid viscosity () and the hydrodynamic nanoparticle volume (Vh) [22]. These two relaxations can be combined to give the effective relaxation time (τeff), eq. 4: 𝜏 𝜏 𝜏𝑒𝑓𝑓 = 𝐵 𝑁 [23] 𝜏𝐵 +𝜏𝑁

Thus the heat dissipation is actually calculated by using the harmonic average of the two relaxations, eq. 5: 2 𝑃 = 𝜇0 𝜒 " 𝑓𝐻𝑎𝑝𝑝𝑙𝑖𝑒𝑑 P is the heat dissipation value which can be replaced in this context by the SAR value. f is the frequency of the applied AMF, Happlied is the amplitude of AMF, µ0 is the permeability



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1,9 1,8

T (°C)

log (SAR)

2,0

1,7 1,6 1,5

2,0

2,1

2,2

2,3

log (H)

-1

SAR (W.g )

120

B

100

f = 471 kHz HAC = 180 G

0

100

14 mM

200

300

140 120 100 80 60 40 20 0

[Fe] = 14 mM

14 mM

Agarose Water

400

500

600

Time (s)

80

Fig. 5. Magnetic hyperthermia: temperature increment during 600 s for MNP-HSA in water at iron concentrations of 14 mM and 28 mM, and immobilized in agarose gel at 14 mM. Inset: SAR at 471 kHz with magnetic field amplitude H = 180 G. Inset (right): panel of images acquired by an IR camera.

60 40 20 100

28 mM

14 mM (Agarose) 14 mM (Water) 28 mM (Water)

8 7 6 5 4 3 2 1 0 -1

-1

A

SAR (W.g )

2,1

200

300

400

500

f (kHz) -3

of free space and χ” is the imaginary part of the AMF susceptibility. The variations of the SAR with the frequency and magnetic field amplitude are depicted in Fig. 4 A and B, respectively. The field amplitude dependence is nearly a quadratic function as showed in eq. 5. This expression was linearized as: log(SAR) = a logH + log(𝜇0 𝜒 " 𝑓) and the following fitting was obtained: log(y) = 1.72(3) log x – 1.81(7) with R2 = 0.998, where a is close to 2, which is in accordance with the Linear Response Theory (LRT model) for superparagmagnetic NPs at this low magnetic field [24], and the dependency on the frequency is linear: y = 0.31(1)x – 23(3) with R2 = 0.996, as expected (Fig. 4A). The temperature increments (T) for MNP-HSA in water are 4.6 and 7.4 °C for iron concentrations of 14 mM and 28 mM, respectively, where a plateau is reached after 500 s of exposure to the magnetic field. The MNP suspension at both concentrations shows good colloidal stability during the magnetic hyperthermia measurements (inset Fig. 5, IR camera images). The related SAR value of 123 ± 2 W.g-1 (Fig. 5) is the same for both concentrations. This value is higher than that reported for iron oxide nanoparticles with diameters between 8 and 11 nm [25]. A value of 120 ± 3 W.g-1 was obtained for MNP-HSA in agarose gel at an iron concentration of 14 mM (inset Fig. 5). Although the viscosity of the gel, η, depends on the average molecular weight, concentration, and type, it can be more than 20 times that of water, high enough for the MNPs to be considered as nearly immobilized [25]. By putting the MNP-HSA nano-platform in a medium of high viscosity, we reduce or suppress the freedom of rotation of the nanoparticles, which leads to a decrease in or to a suppression of heat dissipation by Brownian motion. Thus, since the heat

10

Relaxation Time (s)

Fig. 4. A- Variation of SAR values at 180 G on application of an AMF (144471 kHz). B- Variation of SAR values on application of an AMF at 471 kHz. [Fe] = 28 mM.

 MNP - HSA

 MNP bulk

 MNP - HSA





-4

10



-5

10

-6

10



eff

MNP - HSA

144 kHz

-7

10

471 kHz

-8

10

-9

10

-10

10

0

2

4

6

8 10 12 14 16 18

Nanoparticle diameter (nm)

Fig. 6. Brownian (τB), Néel (τN) and effective (τeff) relaxation times of MNPHSA as a function of the magnetic core diameter.

dissipation, expressed by SAR, is the same as in water, we are led to assume that it is controlled by Néel relaxation. To confirm this fact, theoretical estimates of the variation of Brownian, Néel and effective relaxation times for MNP-HSA with nanoparticle diameter are shown in Fig. 6. Néel relaxation of MNP-HSA was compared to bulk magnetite. The experimental measurement time (τm) is described in the shaded box. In a typical MH study the resonance condition is achieved when 2𝜋𝑓 ∙ 𝜏𝑒𝑓𝑓 = 1. In the case of MNP-HSA this appears at around 10 nm (Fig. 6); at this size the Néel Fig. 5. Brownian (τB), Néel (τN) and effective (τeff) relaxation times of MNPrelaxation process is the faster and dominates τeff. The HSA as a function of the magnetic core diameter. Brownian mechanism is found to dominate at larger nanoparticle diameters, above 14 nm. It must be remembered that the relaxation times calculated here do not take into account the NP size distribution.



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In this work, we show that the mechanism of heat dissipation of 10 nm sized spinel iron oxide single crystals functionalized with HSA (MNP-HSA) occurs through Néel relaxation. This kind of nanoparticle seems to have real potential for moderate magnetic hyperthermia between 41 and 46 °C. The SAR found in this work should be the same in cells since heating is driven only by Néel relaxation. This rise of temperature could affect cells and tissues by inducing permanent irreversible protein damage and regulation of apoptosis. Hyperthermia is receiving more and more attention with these magnetic nanomaterials functionalized by specific proteins in order to involve an intracellular heat to a specified region to treat in the body.

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