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PMIDA-Modified Fe3O4 Magnetic Nanoparticles: Synthesis and Application for Liver MRI Alexander M. Demin, Alexandra G. Pershina, Artem S. Minin, Alexander V. Mekhaev, Vladimir V. Ivanov, Sofiya P. Lezhava, Alexandra A. Zakharova, Iliya V. Byzov, Mikhail A. Uimin, Victor P. Krasnov, and Ludmila M. Ogorodova Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04023 • Publication Date (Web): 24 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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PMIDA-Modified Fe3O4 Magnetic Nanoparticles: Synthesis and Application for Liver MRI *‚

Á†

ˆ

‚

Alexander M. Demin, Alexandra G. Pershina, Artem S. Minin, Alexander V. Mekhaev, Vladimir V. Á Á † ˆ ˆ Ivanov, Sofiya P. Lezhava, Alexandra A. Zakharova, Iliya V. Byzov, Mikhail A. Uimin, Victor P. ‚ Á Krasnov, Ludmila M. Ogorodova ‚

Postovsky Institute of Organic Synthesis of RAS (Ural Branch), 22 S. Kovalevskoy St., 620990, Yekaterinburg, Russia

Á

Siberian State Medical University, 2 Moskovsky trakt, 634050, Tomsk, Russia

†

National Research Tomsk Polytechnic University, 30 Lenina Ave., Tomsk, 634050, Russia

ˆ

Miheev Institute of Metal Physics of RAS (Ural Branch), 18 S. Kovalevskoy St., 620990, Yekaterinburg, Russia

Abstract The surface modification of Fe3O4-based magnetic nanoparticles (MNPs) with N(phosphonomethyl)iminodiacetic acid (PMIDA) was studied and the possibility of their use as MRI contrast agents was shown. The effect of the added PMIDA amount, the reaction temperature and time on the degree of immobilization of this reagent on MNPs and the hydrodynamic characteristics of their aqueous colloidal solutions has been systematically investigated for the first time. It has been shown that the optimum conditions for MNPs modification is the reaction at 40 ƒ& ZLWK an equimolar amount of PMIDA for 3.5 h. The modified MNPs were characterized by X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), infrared spectroscopy (FTIR), thermogravimetric (TGA) and CHN elemental analysis. The dependence of hydrodynamic characteristics of the MNPs colloidal solutions on their concentration and pH of the medium was studied by the dynamic light scattering (DLS) method. Based on obtained data we can assume that PMIDA molecules are fixed on the surface of MNPs as a monomolecular layer. Modified MNPs had good colloidal stability and high magnetic properties. The calculated relaxivities r2 and ±1 ±1 r1 were 341 and 102 mmol s , respectively. The possibility of using colloidal solutions of PMIDA±1 modified MNPs as a T2-contrast agent for liver studies in vivo (at a dose of 0.6 mg kg ) was demonstrated for the first time. Keywords Magnetic nanoparticles, N-(Phosphonomethyl)iminodiacetic resonance imaging, Contrast agent, Liver, Cytotoxicity

*

acid

(PMIDA),

Magnetic

Corresponding author. E-mail: [email protected]; phone: +7 (343) 3623496.


1

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1. Introduction Magnetic nanoparticles (MNPs) of metals (Fe, Co, Ni, Mn), ferrites (MFe 2O4, where M is a divalent 2+

2+

2+

2+

cation of transition metals: Co , Ni , Mg , Zn , etc.) or their oxides (primarily Fe2O3 and Fe3O4) due to their unique physical properties are widely used in modern medicine and biology. Currently, MNPs are often used as diagnostic (magnetic resonance imaging (MRI) or magnetic relaxometry) and therapeutic (laser and magnetic hyperthermia) agents.

1±6

Surface modification of MNPs with biomolecules allows the

preparation of multifunctional agents used as delivery systems for pharmaceuticals, DNA molecules, 7±9

etc.,

biosensors for the diagnostics of various diseases,

10, 11

as well as for molecular and cell

12, 13

separation.

A number of approaches of the non-covalent and covalent modification of MNPs have been used for 1, 14

these purposes.

Although covalent binding of organic molecules to MNPs (for example, using various

alkoxysilane reagents)

2, 15

is the most preferred surface modification method, various amphiphilic

molecules that bind to nanoparticle surfaces in a non-covalent manner are also often used for stabilization 1, 2, 16

and biomedical applications.

Derivatives of carboxylic, sulfonic, phosphoric and phosphonic acids

bind to the MNP surface via non-covalent absorption and allow obtaining stable colloidal solutions of MNPs in aqueous media. Unlike carboxylic acids, there are not many published examples of using phosphonic or phosphoric acid derivatives, although they can be successfully applied to create a hydrophilic, biocompatible and biodegradable coating on MNPs.

17

It is known that phosphonic derivatives form very strong complexes with the surface Fe atoms of nanoparticles due to formation of the P±O±Fe bonds of noncovalent nature. Thus, C. Yee considered two variants of binding of octadecylphosphonic acid to the surface of amorphous Fe 2O3 MNPs involving either one or both OH groups in the formation of a complex with Fe

3+

18

ions on the surface.

In the experiment on

the stabilization of amorphous Fe2O3 MNPs by various acids, K. Shafi et al. have shown that the phosphonate group gives a stronger binding to the MNPs surface compared to the carboxyl or sulfonate groups, probably due to the formation of a bidentate complex with Fe atoms.

19

A stronger binding of

phosphonic derivatives to Fe3O4 MNPs compared with carboxylic acids has also been demonstrated by Y. Sahoo et al.

20

In the above work, dodecyl and hexadecyl phosphonates, as well as dihexadecyl phosphate 21

were used to stabilize Fe3O4 MNPs. Similar reagents were used to stabilize CoFe2O4 MNPs. of phosphates, such as cyclic hexamethophosphate

22

23

and linear sodium triphosphate,

Application

for stabilization of

MNP colloid solutions due to formation of the P±O±Fe bonds was reported by P. Sharma et al. and J. Majeed et al. The amino and carboxyl derivatives of phosphonic acid can be used, on the one hand, to stabilize aqueous suspensions of MNPs, and on the other, for their further covalent surface modification with biomolecules using the methods of peptide chemistry.

24

S. Mohapatra and P. Pramanik studied the

adsorption of carboxymethyl, 2-carboxyethyl, 3-aminopropyl, 4-aminophenyl and 4-carboxyphenyl 25

phosphonic acids on Fe3O4 MNPs in an aqueous medium.

It has been shown that carboxyalkyl

phosphonic acids are attached to the MNP surface via a phosphonic acid residue, since the phosphonate group is a stronger complexone with Fe±O than the carboxyl group. Tudisco et al. reported the synthesis of a preparation that can be selectively loaded with N-methylated drugs or biomolecules due to the presence of tetraphosphonate receptor (Tiiii).

26

Fe3O4 MNPs were surface-modified with 10-

undecynylphosphonic acid, which in this case acted as a bifunctional linker: the phosphonate group provided binding to the MNP surface, and the alkynyl group was used to bind to the azide groups of Tiiii


2

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27, 28

and PEG by the click chemistry methods.

Page 26 of 40

V. Amendola et al. used N-(phosphonomethyl)iminodiacetic

acid (PMIDA) to stabilize FeOx±MNPs obtained by laser ablation in solution.

29

In this case, it has been

shown that PMIDA is the best stabilizer compared to other most common stabilizing agents, such as bovine serum albumin and PEG. PMIDA was used to stabilize MNPs in order to synthesize Fe 3O4@CdS 30, 31

nanohybrid materials based thereof.

PMIDA was also used to prepare multifunctional MNPs as

potential agents for targeted therapy and diagnosis of cancer.

32

An example of designing an agent that

can be used in an immunoassay for cancer diagnostics was reported.

33

The PMIDA-functionalized Fe3O4

MNPs were used to produce nanoparticles with high selectivity for recognizing HER-2 receptors 34

expressed on MCF-7 tumor cells. Another example case was described by Bhattacharya et al. ; MNPs II

II

based on mixed ferrite Mn xFe

III í[Fe 2O4

were functionalized with PMIDA and then conjugated to IgGs

using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). In order to conduct magnetic separation of the 6xHis-containing protein, Sahu et al. obtained PMIDA2+

modified MNPs containing Ni

ions bound by complexation to carboxyl groups.

35

A similar approach using

PMIDA was applied to isolate His-tagged green fluorescent protein and chloramphenicol acetyltransferase from Escherichia coli extracts.

36

In order to design an efficient magnetoseparable biocatalyst, Pramanik et al. performed the conjugation of PMIDA-modified MNPs to urease using EDC.

37

Similarly, the MNPs are modified with bis-phosphonate derivatives, i.e., molecules containing two 38±40

phosphonate fragments.

In order to obtain M13V ZLWK WKH HIIHFW RI ³VWHDOWK´ L H ZKLFK DUH LQYLVLEOH WR

the reticuloendothelial system, Karimi et al. used a number of bis-phosphonates containing quaternary amino groups in their structure.

41

Port et al. and Rerat et al. used the bisphosphonate derivatives to obtain

42, 43

materials for MRI of tumors.

Thus, despite the fact that there are several published examples of using PMIDA to stabilize Fe3O4 MNPs (or to bind any biomolecules on the MNP surface), the modification process with this reagent and the features of its coordination on the MNPs surface have not been systematically investigated. The purpose of the work was to comprehensively study the surface modification of Fe3O4 MNPs (obtained by precipitation from solutions of Fe

3+

and Fe

2+

salts) with PMIDA and to study their magneto-

contrast properties in experiments in vitro and in vivo. The results obtained, we believe, will be useful in the development of MRI-contrast agents, including those for cancer diagnostics. 2. Materials and Methods 2.1. Materials N-(Phosphonomethyl)iminodiacetic acid hydrate (PMIDA, 97%) (Sigma-Aldrich, USA) was used for the syntheses. All other chemicals were purchased from commercial suppliers. These chemicals were of highest purity grade and were used as received. Deionized (DI) water was used as a dispersion medium. 2.2. MNPs surface modification 2.2.1. MNPs synthesis MNPs of a diameter of 11 nm were obtained by co-precipitation from a solution of Fe similar to the previously described procedure.

44, 45

3+

and Fe

2+

salts

Under stirring with an overhead stirrer, 3 mL of a

saturated solution of ammonia was added to a solution of FeSO4î +2O (0.556, 2.0 mmol) and FeCl3î6H2O (1.082 g, 4.0 mmol) in 40 mL of DI water in an ultrasonic bath, to achieve pH 11. The solution was stirred for 20 min at 40 ƒC, precipitated on a magnet and washed with DI water to neutral pH. The MNPs were stored in water suspensions.


3

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2.2.2. PMIDA-modification of MNPs PMIDA-modification of MNPs was carried out in a similar way as described in.

46

A solution of PMIDA

(0.212 mg, 0.864 mmol) in 20 mL of water was added to 60 mL of a suspension of MNPs (0.200 g, 0.864 mmol) under stirring in an ultrasonic bath. The reaction mixture was stirred in an ultrasonic bath for 3 h, and then the resulting suspension was centrifuged at 17,000 rpm for 20 min to remove excess PMIDA. The precipitate was washed several times with DI water to neutral pH of supernant and resuspended in DI water to afford suspensions of the PMIDA-modified MNPs. 2.2.3 MNPs characterization X-Ray diffraction (XRD) measurements were evaluated using a Shimadzu XRD-6000 X-ray diffractometer (Japan, Cu Ka-radiation). Transmission electron microscopy (TEM) images of the MNPs were obtained on a Philips CM30 scanning transmission electron microscope. The IR spectra were recorded on a Nicolet 6700 FTIR-spectrometer (Thermo) by the ATR method on í1

í1

the diamond crystal in the range of 4000±400 cm with 192 scans and at 4 cm resolution. The mass fraction of carbon was measured using a &+1 J?

,, automatic analyser

(PerkinElmer Inc., Waltham, MA, USA). The amount of PMIDA residues immobilized on the MNP surface was calculated by the equation: k = Z1 * 1000 , Z2 * M where k is the amount of PMIDA on the surface of nanoparticles, mmol per 1 g of MNPs; &1 is the carbon mass fraction of the MNPs-COOH sample; &2 is the calculated carbon mass fraction in the residue of PMIDA; ±1

F is the molecular weight of PMIDA (227.11 g mol ). Thermogravimetric analysis (TGA) was performed on ~5 mg of sample using a TGA/DSC1 thermogravimetric analyzer (Mettler Toledo, Columbus, OH, USA) with a heating rate of 10 ƒ& PLQ a temperature range of 30 ƒ&±900 ƒ& XQGHU FRPSUHVVHG $U

í

over

í

P/ min ).

To compare the TGA data on the PMIDA amount on the MNP surface with the elemental analysis í1

data, the weight loss of the samples (%) was converted into units of mmol g

by the equation:

k = Z1 *1000 , M where k is the amount of PMIDA on the surface of nanoparticles, mmol per 1 g of MNPs; &1 is the weight loss of the MNPs-COOH sample (mass fraction); -1

F is the molecular weight of PMIDA (227.11 g mol ). Zeta potential (zP) and dynamic light scattering characterization were carried out using Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The specific magnetization of the powders of modified MNPs was measured at room temperature ±1

using a vibration magnetometer in a magnetic field up to 2.2 MA m . The concentration of iron in the suspension of MNPs for in vitro and in vivo experiments was measured by atomic emission spectrometry with inductively coupled plasma (iCAP 6300 Duo; Thermo Fisher Scientific, Waltham, MA, USA) and magnetometric method (Faraday balance). The resulting ferromagnetic contribution value was used to calculate the proportion of magnetic cores (ferrite) corresponding to nanoparticles contained in the suspension.


4

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Page 28 of 40

2.3. FRI experiments The relaxivity of the PMIDA-modified MNPs dispersed in water (with the [Fe] concentration ranging IURP

P0 WR

—0 ZDV GHWHUPLQHG XVLQJ D UHOD[RPHWHU (&27(. &RUSRUDWLRQ +RXVWRQ 7;

USA). The T2 measurements were performed using the corresponding Carr-Purcell-Meiboom-Gill pulse sequence (

ƒ±echo time [TE]/2±

ƒ±echo) (4.31 MHz, echo 1 ms). To measure T1, a modified

saturation recovery pulse sequence (repetition time [TR]i±

ƒ±TE/2±

ƒ±echo, where TRi is the ith value

of the time recovery) was used. Relaxivity data were expressed as a slope of the relaxation rate vs concentration. The slope was calculated by best-fit linear regression analysis using the least squares method. 2.4. MRI in vivo For in vivo experiment, 10-week-old male golden hamsters (Mesocricetus auratus) were used. Hamsters were housed three to a cage (OptiRAT) under conventional conditions and were fed with stock diet and water ad libitum. The animals were handled according to the regulations of the Animal Care and Use Committee of the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, and kept in a pathogen-free environment. The animals were anaesthetized with gas anesthesia (Isofluran; Baxter Healthcare Corp., Deerfield, IL) using a Univentor 400 Anesthesia Unit (Univentor, Zejtun, Malta). During in vivo H[SHULPHQWV

—/ RI

P0 >)H@ 013V VXVSHQVLRQ LQ SKRVSKDWH-buffered saline (PBS;

137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) was administered retro-orbital. High-resolution T2-weighted images of the hamster liver were recorded on an 11.7 T horizontal MRI scanner (Biospec 117/16 USR; Bruker Optik GmbH, Ettlingen, Germany) equipped with a receiver± transmitter 1H volume coil (T11440V3) and acquired with respiratory triggering using L2 RARE (Rapid Imaging with Refocused Echoes) method with pulse sequence parameters: TEeff = 7.5 ms, TR = 1500 ms, )OLS $QJOH

ƒ

After MRI scanning for 1 h, animals were sacrificed; the liver was extracted and placed in 10% neutral buffered formalin for histological analysis. Standard haematoxylin and eosin and Prussian blue staining protocols were used. The histological analysis was performed using optical microscope Axiostar Plus (Carl Zeiss Meditec AG, Jena, Germany). The experimental protocol has been approved by the Bioethics Review Committee of the Institute of Cytology and Genetics (No 24 from October 28, 2014). 2.5. In vitro cytotoxicity evaluation MNPs in five concentrations (5±

í

—J mL ) were incubated with CHO and MDA-MB231 cell lines

for 24 h. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 47

(MTT) assay and using Annexin V/7-aminoactinomycin D kit (BD Biosciences, San Jose, CA, USA) on a BD Accuri-C6 flow cytometer. Cell viability expressed as a fraction of viable cells normalized to the cells without co-incubation with MNPs (blank control). 3. Results and discussion 3.1. MNPs surface modification and characterization Among the phosphonic acid derivatives used to stabilize colloidal solutions of MNPs, the most interesting are derivatives containing any additional functional groups other than the phosphonate group, which allows further modification of MNPs. Thus, PMIDA forms the sufficiently strong P±O±Fe bond of a non-covalent nature with the surface Fe atoms of MNPs and, being inherently hydrophilic molecule, can be used to stabilize their aqueous suspensions or for further functionalization. Although there are several


5

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Langmuir

published examples of the use of PMIDA for this purpose,

29±37

the surface modification of MNPs by this

reagent has not been systematically investigated. In the present study, we used MNPs obtained by precipitation from a solution of Fe

3+

and Fe

2+

salts

with an average particle diameter of 11 nm, which were Fe3O4 magnetite in its phase composition, as 44-46

confirmed by XRD and TEM data (Fig. 1).

Functionalization of the surface of magnetic nanoparticles with PMIDA was carried out in accordance with Scheme 1.

Scheme 1. Functionalization of the MNP surface with PMIDA (schematic image). According to the XRD measurements and TEM data, significant alterations of the structure of nanoparticles did not occur during modification. The observed diffraction peaks in XRD patterns (Fig. 1a) and in the selected area of electron diffraction patterns (Fig. 1b, c) of bare MNPs and MNPs-COOH coincide with the Jade database (JCPDS Card No. 85-1436), indicating that the cores of these MNPs have a cubic inverse spinel Fe3O4 structure with reflection planes (220), (311), (400), (422), (511) and (440).

a

b

c

Fig. 1. (a) XRD patterns, (b) TEM images and electron diffraction patterns (inset boxes) of MNPs and (c) MNPs-COOH. In first stage we studied the influence of the amount of PMIDA used in reaction on the loaded level of PMIDA on MNPs. To do this, 0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 1.5, and 3.0 molar excess of PMIDA relative to Fe3O4 (or 0.2, 0.4, 1.0, 2.0, 3.0, 4.0, 6.0, and 12.0 mmol PMIDA per 1 g of MNPs, respectively) were -1

added to an aqueous suspension of MNPs (2 mg mL ). The reaction was carried out under stirring for 16 h. The immobilization was confirmed by the IR data (Fig. 2). In the FTIR spectra of PMIDA-modified MNPs, absorption bands characteristic of both the Fe3O4 ±1

nanoparticles (stretching vibrational mode of Fe±O, 535 cm ) and phosphonomethyl derivatives (C=O, 2±

RPO3 , CH2) are observed. Thus, the individual narrow bands in 1300±900 cm


±1

region (Fig. 2, PMIDA) 6

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Langmuir

-1

A concentration range of 0.1±2.8 mg mL in which Dh is maintained at an average of 61 nm (PdI 0.20) and zP is an average of ±23.5 mV was found. Dh increases insignificantly at high concentrations of MNPs. -1

While under strong dilutions (at concentrations below 0.1 mg mL ) we observed a change in Dh to 123 nm and zP to ±32 mV. Similar concentration effects on Dh of nanoparticles in suspension were reported for 3aminopropylsilane-modified nanoparticles.

47

Fe3O4

MNPs,

44

multi-walled

carbon

nanotubes,

silica

and

gold

Observed in our work the increase in the particle size can be explained by an increase in

the contribution of the signal from extraneous particulate matter or by the nature of the homodyne optical configuration of used equipment. We also studied how the hydrodynamic characteristics of MNP colloid solution depend on pH (jG 2±12) (Fig. 6b). High stability of colloid solutions of MNPs has been demonstrated in the pH range from 3 to 12 (Dh remained constant, 55 nm (PdI 0.24)). It has been shown that the charge exchange of the surface of magnetic nanoparticles occurs at pH 2 (zP 0.8 mV), the maximum zP (±55 mV) was reached at pH above 11 due to the dissociation of both PMIDA carboxyl groups on the surface of magnetic nanoparticles. 3.2. FRI experiments During the work the magnetic properties of PMIDA-modified MNPs were studied (Fig. 7a,b). An increase in the amount of PMIDA on the MNP surface naturally leads to a slight decrease in the value of ±1

the saturation magnetization (M) in comparison with that of the initial MNPs (75 emu g ) (Fig.7b). It was -1

shown that M of PMIDA-modified MNPs containing 0.50-0.65 mmol g PMIDA on the surface had close ±1

values (64 emu g ). The magnet materials used as contrast agents provide the shortening of the T2-weightened relaxation time. Consequently the decreasing of relaxation time of the tissues containing MNPs is appeared in MRI images as a blackout of the corresponding areas. The level with contrast agent influence on relaxation time is characterized by relaxivity coefficient (r2). Therefore, the relaxivity r2 and r1 of colloidal solutions with MNPs-COOH concentrations from 1.07 to ±4

1.05 10

mM (11 points used) was measured, at which the dependence of R 1 and R2 on [Fe]

concentration are linear (standard deviation 0.9998 and 1.0000 for R2 and R1, respectively) (Fig. 7b). Thus, r2 was 341.2; r1, 101.8 mmol

±1

s

±1

(r2/r1 3.35), which exceeds the data for some commercial

agents, for example, Feridex (r2 98.3 and r1 23.9 mmol ±1

mmol

s

±1

±1

s

±1

(r2/r1 4.11)), Resovist (r2 151.0 and r1 25.4 ±1

(r2/r1 5.94)), and Lumirem (r2 72 and r1 3.2 mmol

s

±1

(r2/r1 22.5)).

48

However, such high

value of r2 is in the limits of relaxivity coefficient values reported for Fe 3O4 MNPs.

49

For example, for

MNPs coated with Polyethylenimine 2k and dispersed in water as nanoclusters (Dh 55 nm), r2 was 345 ±1

mmol

±1

s . The r2 value decreased to 121 (Dh ~20 nm) and 84 mmol

±1

s

±1

(Dh

of 40 ACS Paragon Plus Environment Langmuir 1 2 3 4 5 6 7 8 9 10

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