Octadecylamine-Mediated Versatile Coating of CoFe2O4 NPs for the

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Octadecylamine Mediated Versatile Coating of CoFe2O4 NPs for the Sustained Release of Anti-inflammatory Drug Naproxen and in vivo Target Selectivity Violetta Georgiadou, George Makris, Dionysia Papagiannopoulou, Georgios Vourlias, and Catherine Dendrinou-Samara ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b00408 • Publication Date (Web): 17 Mar 2016 Downloaded from http://pubs.acs.org on March 19, 2016

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ACS Applied Materials & Interfaces

Octadecylamine Mediated Versatile Coating of CoFe2O4 NPs for the Sustained Release of Anti-inflammatory Drug Naproxen and in vivo Target Selectivity Violetta Georgiadou,1 George Makris,2 Dionysia Papagiannopoulou2, Georgios Vourlias3 and Catherine Dendrinou-Samara1* 1

Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki,

Greece 2

Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of

Thessaloniki, 54124 Thessaloniki, Greece 3

Department of Physics, Aristotle University of Thessaloniki, 54124 Thessaloniki,

Greece * Corresponding author, Email: [email protected], Tel: +30-2310-99-7876

Abstract Magnetic nanoparticles (MNPs) can play a distinct role in magnetic drug delivery via their distribution to the targeted area. The preparation of such MNPs is a challenging multiplex task that requires the optimization of size, magnetic and surface properties for the achievement of desirable target selectivity, while the sustained drug release is a prerequisite.

In that context, CoFe2O4 MNPs of small size ~7 nm and moderate

saturation magnetization ~60 emu g-1 were solvothermally synthesized in the presence of octadecylamine (ODA) with a view to investigate the functionalization route effect on the drug release.

Synthetic regulations allowed us to prepare MNPs with aminated

(AmMNPs) and amine-free (FAmMNPs) surface. The addition of the non-steroidal antiinflammatory drug with a carboxylate donor, Naproxen (NAP) was achieved by direct coupling with the NH2 groups, rendered by ODA, through the formation of an amide bond in the case of AmMNPs. In case of FAmMNPs indirect coupling of NAP was performed through an intermediate linker (polyethyleneimine) and on PEG-ylated MNPs. FT-IR, 1H-NMR,

13

C-NMR and UV-Vis data confirmed the addition of NAP, while

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diverse drug release behavior was observed for the different functionalization approaches. The biological behavior of the MNPs@NAP was evaluated in vitro in rat serum and in vivo in mice, after radiolabeling with a γ-emitting radionuclide, 99mTc. The in vivo fate of MNPs@NAP carriers was in straightforward relation with the direct or indirect coupling of NAP. Furthermore, an inflammation was induced intramuscularly, where the directly coupled 99mTc-MNPs@NAP carriers showed increased accumulation at the inflammation site.

Keywords Magnetic nanoparticles• anti-inflammatory drug-carriers• controlled drug release• technetium-99m • biodistribution

Abbreviations MNPs, magnetic nanoparticles; NAP, naproxen; ODA, octadecylamine; DPE, diphenylether; PEI, polyethyleneimine; PC, paper chromatography; RCY, radiochemical yield; p.i., post injection.

1. Introduction Most of non-steroidal anti-inflammatory drugs (NSAIDs) show chemopreventive and antitumorigenic activity by reducing the number and size of carcinogen-induced colon tumors and by enhancing the efficacy of antitumor drugs.1 The main role of NSAIDs is to block the formation of prostaglandins with the inhibition of the enzymes responsible of their production, (cyclooxygenases, COX). However, long-term reception of NSAIDs weakens their activity owing to gastrointestinal and renal side effects due to a combination of local irritations produced by their free carboxyl group and by suppression of the cytoprotective prostaglandins on gastric mucosa.2 Magnetic nanoparticles (MNPs) offer a bimodal and/or multiple capabilities in clinical use3, due to the ability to perform as contrast agents in magnetic resonance imaging and/or in magnetic hyperthermia while they are proposed as drug carriers in order to surpass the limitations that drugs undergo due to poor targeting or cytotoxicity and decreased efficacy. To our knowledge, the use of MNPs as NSAIDs carriers has not 2 ACS Paragon Plus Environment

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been given much attention. Recently, CoFe2O4 MNPs are under investigation as potential drug carriers for targeted drug delivery4,5 and they could be therefore considered for encountering the drawbacks of the NSAIDs’ clinical behavior by improving the efficiency and attenuating the side effects. A considerable shortcoming of the extensive use of MNPs is their potential cytotoxicity. Generally, in order to overcome this problem well protected MNPs and in low concentrations have to be used. Regarding their cytotoxicity, it is determined by several factors including both cell physiology and MNPs properties such as size, material, surface characteristics and shape as well.6,7 The design of “smart” MNPs carriers aims to the selective targeting of biological entities by avoiding the biological constraints inside the body (“in vivo barriers”) that protect the body against external species intrusion. These “in vivo barriers” that MNPs come across when entering the body, limit their movement, cause modifications to the magnetic and surface properties, or induce a negative host response using biochemical signaling and thus can result in an early uptake by cells before the MNPs manage to reach the target tissue.8 For example, blood can cause MNPs to agglomerate when entering the body through intravascular administration. Till recently, indirect drug addition has been studied in depth through intermediate biocompatible coatings e.g. Polyethyleneglycol (PEG) co-polymers, Polyethyleneimine (PEI) molecules, dextranes, liposomes, etc. applied on as prepared MNPs.8 On the other hand, the direct drug coupling is a complicated issue, that requires as prepared MNPs with specific surface features for the covalent addition of the drug on their coating. Therefore, the preparation of MNPs for drug delivery is a task with many challenges, including suitable surface, size and magnetic properties as prerequisites for a desirable biodistribution/target selectivity and sustained drug release.9 Herein, we describe the synthesis of fine cobalt ferrite magnetic nanoparticles (CoFe2O4 MNPs) in the presence of octadecylamine (ODA) and how simple synthetic variations affect the surface properties of the organic coating of MNPs and further advance their use as drug carriers. The presented synthesis is designed with a view to facilitate the post synthesis functionalization, considering that these processes, in most of the studies, involve complicated procedures. Indeed, when ODA was used in a triple role (coating, solvent, reducing agent), CoFe2O4 MNPs with aminated surface (AmMNPs) 3 ACS Paragon Plus Environment


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were yielded; while the presence of a non polar solvent (diphenylether, DPE) in the preparation led to the isolation of CoFe2O4 MNPs with amine free surface (FAmMNPs). Based on the different surface properties of the resulting CoFe2O4 MNPs, the attachment of Naproxen (NAP), a carboxylate NSAID, commonly used to treat the inflammation and pain of various wounds10 has been facilitated. Three different pathways were employed for the preparation of CoFe2O4 MNPs@NAP carriers in order to be compared: i) direct coupling through the ODA amino groups; ii) indirect coupling through the amino groups of the intermediate linker PEI and iii) loading on a PEG matrix after the PEG-ylation of the MNPs. The functionalized MNPs@NAP that demonstrated sustained drug release were further examined in vivo. The biological evaluation of the MNPs was performed after radiolabeling with

99m

Tc.

99m

Tc-radiolabeling was selected as a technique that does

not alter the MNPs’ properties, in order to monitor/assess their biodistribution profile in mice. Technetium-99m is a widely used imaging agent in nuclear medicine that emits γ radiation and has also been used for the development of nanoparticle-based radiopharmaceuticals.11-13 The biodistribution studies revealed that the target selectivity of the NAP carriers CoFe2O4 MNPs towards the inflammation is in direct relation with the functionalization method that was followed for the addition of NAP.

2. Materials and Methods Materials All the reagents were used without any further purification. Iron (III) acetylacetonate (Fe(acac)3), octadecylamine (>90.0%) and N,N-Dimethyl formamide (>99.5%) were purchased by Fluka, cobalt (III) acetylacetonate (Co(acac)3, ≥99.9%) was supplied by E. Merck, AG. Darmstaadt, cobalt (II) acetylacetonate (Co(acac)2, ≥99.9%) was from Baker Chemicals, CDCl3 (99.9%) was purchased by Deutero GmbH, ethanol (100%, 1% MEK) was from Bruggermann GmbH. Chloroform (analytical reagent) were from Chem. Lab NV. Diphenylether (DPE), ninhydrin (GR for analysis ACS, Reag. Ph Eur) were purchased by Merck. The reagents used for the BaCl2/I2 assay were: I2 (May &Baker LTD), KI (Merck), starch (Sigma Aldrich), BaCl2 (Merck). Phospate buffer pH 6.4 was prepared with NaH2PO4 (Merck), Na2HPO4 (Merck) and NaCl (Merck). 1-(3Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, (EDC, >98.0 %, coupling 4 ACS Paragon Plus Environment

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agent for peptides) was purchased from TCI (Tokyo Chemical Industry) and PEG-8000 from Alfa Aesar GmbH & CoKG. Triethylamine (Et3N, ≥99.5%) and Nhydroxysuccinimide (NHS, 98.0%) were from Sigma-Aldrich and (S)-(+)-2-(6-methoxy2-naphthyl)propionic acid (Naproxen) and poly(ethyleneimine) solution (PEI, Mn ~1,800) from Aldrich Chemistry. Deionized water was used for the fabrication of CoFe2O4 MNPs and Milli pure water (< 3.0 MΩ, Millipore, MilliQ Gradient) was used for the modification of the NPs surface properties. Nanofilters Minisart RC 15 (Single use syringe filter Non-sterile RC-membrane, Pore Size: 0.20 µm) were from Sartorius Stedim Biotech GmbH. Pur-A-Lyzer Midi 1000 Dialysis Kit (for Research & Development use only) by Sigma-Aldrich. Na99mTcO4 was obtained from a commercial 99

Mo/99mTc

generator

measurements of

from

A.H.E.P.A.

Hospital,

Thessaloniki.

Radioactivity

99m

Tc samples were done in a dose calibrator (ATOMLABΤΜ 100,

Biodex Medical System) and in a NaI(Tl) scintillator (Caprac®, Capintec).

Characterization Techniques Powder X-ray diffraction (XRD) was performed using a 2-cycle Rigaku Ultima + diffractometer (40 kV, 30 mA, CuKa radiation) with Bragg-Brentano geometry (detection limit 2% approximately). Conventional TEM images were obtained with a JEOL 100 CX microscope (TEM); stable dispersions of the NPs in toluene were prepared for transmission electron microscopy. Energy dispersive detector (EDS) integrated to a scanning electron microscopy instrument (SEM), JEOL 840A and an inductively coupled plasma optical emission spectroscope (ICP-OES, ICP Simultané VARIAN Vista Axial) were used for elemental analysis. Fourier transform infrared spectroscopy (280-4000 cm1

) was recorded using a Nicolet FTIR 6700 spectrometer with samples prepared as KBr

pellets. Thermogravimetric analysis (TGA) was performed using a SETA-RAM SetSys1200 instrument at a heating rate of 10 °C min-1 under N2 atmosphere.

Magnetic

measurements were acquired by a superconducting quantum interference device (Quantum Design MPMS-5 SQUID) and a vibrating sample magnetometer (1.2H/CF/ HT Oxford Instruments VSM). 1H-NMR and

13

C-NMR spectra of the isolated organic

coatings of the samples were received in CDCl3 with TMS (500 MHz, Agilent Technologies); the isolation of the organic coating was achieved after the dissolution of 5 ACS Paragon Plus Environment


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the samples in CHCl3 and several cycles of sonication and centrifugation, until the supernatant was limpid. The supernatant from each washing cycle was removed and placed into glass vials. Then it was filtered with the use of nanofilters to retain the dispersed NPs. The resulting pale yellow liquids were condensed, and CDCl3+TMS was added for their further spectroscopic studies. UV-Visible measurements were carried out with a double beam UV-visible spectrophotometer U-2001 Hitachi. The DLS measurements were performed on a Malvern Zetasizer instrument.

Preparation of CoFe2O4 MNPs with aminated surface (AmMNPs) CoFe2O4 MNPs with aminated surface (AmMNPs) were prepared in an autoclave by the decomposition of acetylacetonate iron (III) and cobalt (III) at a 2:1 ratio, Fe(acac)3 1.8 mmol : Co(acac)3 0.9 mmol in the presence solely of ODA 12.9 mmol. The temperature of the oven was elevated with a steady rate (4 °C/min) to 200 °C and was kept stable for 24 h. After the 24 h reaction the autoclaves were left to cool down to room temperature with a rate of 5 °C/min and CoFe2O4 MNPs were isolated after repeated washing cycles with EtOH and centrifugation (5000 rpm).

Preparation of CoFe2O4 MNPs with amine-free surface (FAmMNPs) CoFe2O4 MNPs with amine-free surface, samples FAmMNPs1, FAmMNPs2 and FAmMNPs3, were prepared in an autoclave by the decomposition of acetylacetonate iron (III) and cobalt (III) or cobalt (II) in different ratios, while in all preparations the amount of ODA (0.998 mmol) and DPE (7 mL) was constant. Thus, for sample FAmMNPs1, Fe(acac)3 0.9 mmol : Co(acac)3 0.9 mmol ratio was used, while Fe(acac)3 1.8 mmol : Co(acac)3 0.9 mmol and Fe(acac)3 1.8 mmol : Co(acac)2 0.9 mmol were used for samples FAmMNPs2 and FAmMNPs3 respectively. The temperature of the oven was elevated with a steady rate (4 °C/min) to 200 °C and remained stable for 24 h. After the 24 h reaction the autoclaves were left to cool down to room temperature with a rate of 5 °C/min and CoFe2O4 MNPs were isolated after repeated washing cycles with EtOH and centrifugation (5000 rpm).

Ninhydrin Colorimetric Assay 6 ACS Paragon Plus Environment

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Aliquots of 0.1-0.6 mL of ODA (0.25 mg mL-1) in DMF were pipetted into a series of tubes.14 0.7 mL of ninhydrin solution in MeOH 0.06 M (10.7 mg mL-1) were added in each tube, mixed well and heated in a water bath at 100 °C for 5 min. After a short heating period (5 min) the color of the solution changed (formation of Ruhemann’s purple) and the content was transferred to a 5 mL volumetric flask and was diluted with DMF for the UV-Vis absorbance measurement. A suspension of MNPs in DMF (0.25 mg mL-1, stock) was prepared accordingly for all the samples (AmMNPs, FAmMNPs1-3) and 0.4 mL of each stock solution was pipetted into boiling tubes with 0.7 mL of ninhydrin solution following the same procedure described above. The absorbance of the formed complex (Ruhemann’s purple) was recorded at ~600 nm.

Direct Coupling with Naproxen on MNPs with aminated surface The direct coupling of NAP onto the AmMNPs surface coating was achieved via a modified procedure,15 where 20 mg of AmMNPs were mixed with EDC (47.9 mg), NHS (~3 mg) and NAP (57.5 mg), Et3N (0.1 mL) in CHCl3 (5 mL). The mixture was shaken vigorously for 24 h and the AmMNPs-NAP was isolated after repeated washing cycles with EtOH and centrifugation.

Indirect Coupling of Naproxen on MNPs with amine-free surface For the indirect coupling of NAP with the FAmMNPs3, a two-step procedure was followed. Firstly, 5 mL of FAmMNPs3 in CHCl3 solution (2 mg mL-1) and 5 mL of PEI in H2O solution (200 mg mL-1) were prepared. The two solutions were mixed together and the new resulting mixture was shaken vigorously for 3 h. After 3 h of shaking the hydrophobic MNPs were transferred into the water phase and CHCl3 was evaporated from the system.

The FAmMNPs3 coated with PEI (~1,8

kDa), namely

FAmMNPs3@PEI was isolated after washing cycles with EtOH and centrifugation. The resulting powder was characterized and the presence of the extra coating surrounding the MNPs was confirmed. During the second step the FAmMNPs3@PEI was suspended in Η2Ο (3 mL) and was mixed with a solution of EDC (47.9 mg), NHS (~3 mg), NAP (57.5 mg) and Et3N (0.1 mL) in CHCl3 (5 mL). The mixture was shaken for 24 h and the



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functionalized FAmMNPs3@PEI-NAP was isolated after the removal of the water phase and repeating washing cycles with EtOH.

Loading of Naproxen on MNPs with amine-free surface A 3 mL solution of FAmMNPs3 in CHCl3 (5 mg mL-1) was added drop-wise in a 25 mL solution of PEG-8000 in H2O (3.2 mg mL-1), the two-phase mixture was sonicated in a cold sonication bath (25-30 °C) for ~2 h until a stable emulsion was formed (high temperature prevents the formation of a stable suspension). After ~2 h, 40 mg of PEG8000 were added in the emulsion and sonication was applied for 30 min. The CHCl3 was evaporated from the system with elevation of temperature of the sonication bath and then the solution was left to cool to room temperature. 120 mg of PEG-8000 were added in the solution anew followed by the addition of 2 mL of NAP solution in CHCl3 (5 mg mL-1). The emulsion was sonicated again for 2 h and CHCl3 was evaporated. The FAmMNPs3@PEG-NAP was isolated through washing and centrifugation cycles with EtOH and was dried under vacuum.

Determination of PEG-8000 amount on MNPs@PEG-NAP by BaCl2/I2 assay An adapted barium iodide assay was used,16,17 for the characterization of PEG8000 amount on FAmMNPs3@PEG-NAP, A calibration curve was made for a series of PEG-8000 solutions in H2O (50, 40, 30, 20, 15, 10, 5 µΜ). 1 mL of each solution was mixed with freshly prepared 0.25 mL BaCl2 (5 % w/v) and 0.125 mL I2 (0.1 M) solutions and reacted for 15 min (vortex). After the 15 min reaction a red precipitate was formed; the mixtures were filtered and the absorbance of the supernatant was measured at ~535 nm. Accordingly, 1 mL of FAmMNPs3@PEG-NAP aqueous dispersion (0.085 mg mL-1) was mixed with 0.25 mL BaCl2 (5 % w/v) and 0.125 mL I2 (0.1 M) and reacted for 15 min. The dispersion was filtered and the supernatant was diluted to 10 mL and was measured at 535 nm.

Drug Release For the drug release study, ~3 mg of the functionalized MNPs were placed into a Pur-A-Lyzer Midi 1000 dialysis membrane. The membrane floated in phosphate buffer 8 ACS Paragon Plus Environment

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solution (PBS, 45 mL, pH 6.4) and was shaken (200 rpm) at 35 °C. In certain time intervals 2 mL of the medium were removed and replaced with fresh PBS solution. This procedure

was

followed

for

NAP

(control,

Figure

S7),

AmMNPs-NAP,

FAmMNPs3@PEI-NAP and FAmMNPs3@PEG-NAP. UV measurements were recorded at 265 nm and the cumulative release in percentage (Q %) was calculated and plotted against time as the mean of 3 values for each sample according to Equation 110, 18:     A × V   ((A + A + .....) × R )    n −1 n −2   n × C     +           A s   As   × 100, Eq.1 Qn % =    W      

where, A1 is the absorption of the first medium removal (PBS pH 6.4), A2 the absorption of the second medium removal (PBS pH 6.4), An is the absorption of the nth medium removal (PB pH 6.4), As (absorption of the standard) for each sample, determined by the calibration curve of NAP in PBS, pH 6.4 (Figure S8), V is the total volume of the medium (mL), C concentration of the standard (mg mL-1), W weight of the active compound (mg (label claim)), R removed medium volume (mL) and n is the removal number. The NAP release profiles were estimated by using Ritger-Peppas equation (Eq. 19, 20, 21

2)

Q = ktn

Eq. 2

where Q is the cumulative drug release in percentage, t is the release time in hours, k is a rate constant reflecting the structural and geometric characteristics of the carriers, and n is the release component that corresponds to the release mechanism of the drug.

Radiolabeling The

radiolabeling

of

four

samples:

AmMNPs,

AmMNPs-NAP,

FAmMNPs3@PEI and FAmMNPs3@PEI-NAP was performed according to literature procedures12, 22: A solution of 250 µL Na99mTcO4 (5-10 mCi) in saline and 50 µL SnCl2 (1 mg/mL in 0.1 N HCl) was mixed with a dispersion of 250 µL MNPs in PBS (metal content 0.4 % w/w). The labeling mixture was incubated at room temperature (25 oC) for 1 h with periodic vortexing and bath sonication. The

99m

Tc-MNPs were separated via a 9

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magnet, the labeling mixture was removed, and then the 99mTc-MNPs were washed twice with 250 µL PBS and were dispersed in 500 µL PBS. The % radiochemical yield (RCY) was calculated as follows by using a γ-counter: % RCY = 100*(radioactivity of

99m

Tc-

MNPs)/(total radioactivity). Quality control was performed by ascending paper chromatography (PC) either using saline or acetone as the mobile phase (in triplicates). Each paper strip was then cut in three pieces: i) Rf=0-0.1, ii) Rf= 0.1-0.9 and iii) Rf=0.9-1 and the radioactivity of the pieces was measured in a γ-counter. calculated by PC/saline where

99m

Tc-MNPs purity was

99m

Tc-MNPs stays at the origin (Rf=0-0.1) and

pertechnetate (99mTcO4-) as impurity was calculated by PC/acetone where it migrates to the front (Rf=0.9-1), using the formulas: % 99mTc-MNPs = 100*(radioactivity at Rf=0-0.1 in PC/saline)/(Total radioactivity in PC/saline) and %

99m

TcO4-=100*(radioactivity at

Rf=0.9-1 in PC/acetone)/(Total radioactivity in PC/acetone). Stability Studies The stability of

99m

Tc-MNPs was determined by incubating dispersions of the

99m

Tc-MNPs (50 µL) in: a) PBS buffer (0.5 mL, pH 7.4) at room temperature and b) rat

serum (0.5 mL) at 37 oC, for a period of 24 h. Aliquots of dispersions A and B were analyzed at 1, 3, 24 h by PC using either saline or acetone as mobile phase. The % 99mTcMNPs stability and the % pertechnetate were calculated by the formulas described above. Serum stability was measured at 1, 3, 24 h as follows: the

99m

Tc-MNPs were separated

from the serum and their radioactivity was measured in a γ-counter and then an aliquot of the serum was analyzed by PC either in saline or acetone as described above. The % 99m

Tc-MNPs

stability

100*(radioactivity of

was

calculated

by

the

formula:

%

99m

Tc-MNPs

=

99m

Tc-MNPs collected by the magnet)/(Total radioactivity

measured in serum). The percent of pertechnetate was calculated as described above.

Biodistribution studies The biodistribution experiments were approved by the Aristotle University Committee for Animal Experimentation, according to the EU guidelines. Inflammation edema was induced in male BALB/c mice, of approx. 25 g weight by intramuscular injection of 2% carrageenan (0.1 mL) in the right hind limb 2 h prior to the experiment. 10 ACS Paragon Plus Environment

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Each animal was then injected intravenously, each with 10 µCi of the purified

99m

Tc-

MNPs dispersion in saline (100 µL, approximately 6 µg of [Fe+Co] metal content) or Na99mTcO4. Animals (three in each group) were euthanized 1 h and 24 h p.i. by cervical dislocation followed by blood withdrawal and cardiectomy. Organs and tissues of interest were excised rapidly, weighed, and their radioactivity was determined using a γ-counter. The activity of the tissue samples was decay-corrected and calibrated by comparing the counts in the tissue with the counts of a standard solution corresponding to 1% of the injected dose. Counts of the sample and calibration aliquots were measured in the γcounter at the same time. The amount of activity in the selected tissues and organs is expressed as a percent of the injected dose per gram tissue (% ID/g). Values are quoted as the mean % ID ± standard deviation (SD) of the three mice per group. Blood volume and muscle mass were estimated at 7 and 43 % of body weight, respectively.

3. Results and Discussion 3.1. Synthetic Aspects and Characterization of MNPs In continuing our previous efforts14, 23 we selected ODA as a surfactant for the solvothermal preparation of CoFe2O4 MNPs due to its ability, under specific synthetic conditions, to render free NH2 groups on the surface of the MNPs (aminated MNPs) that can serve for further functionalization. In so, when Fe(acac)3 and Co(acac)3 (2:1) were used as precursors and solely ODA in a triple role, aminated CoFe2O4 MNPs were prepared (AmMNPs). The decomposition of the same precursors and ratio with an aprotic solvent (DPE) in the presence of ODA as a surfactant resulted to amine-free CoFe2O4 MNPs with an impurity of CoO phase (FAmMNPs2). Meanwhile, pure CoFe2O4 MNPs were isolated when the equimolar mixture of Fe(acac)3 and Co(acac)3 (sample FAmMNPs1) and the non-equimolar mixture of Fe(acac)3 and Co(acac)2 (2:1) (sample FAmMNPs3) were employed. ODA prompted the nucleation and growth process of the nanomaterials and prevented aggregation phenomena, proving to be a crucial agent in the synthesis of ferrite nanoparticles as shown by others and us.24,25



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1 2 3 Table 1. Characterization of isolated CoFe2O4 MNPs. 4 5 Samples prepared in ODA 6 7 size lattice size Ms 8 Hc TG Ms TB 9 sample XRD parameter TEM corrected formula (% ) (emu/g) (Oe) (K) 10 (emu/g) (nm) (Å) (nm) 11 AmMNPs 10.0 8.420 36 7.5 28.5* 45.0 222 >300 Co1.10Fe1.90O4 12 13 14 Samples prepared in ODA/DPE 15 16 FAmMNPs1 5.50 8.396 46 3.0 23.0** 43.0 127 Co1.26Fe1.74O4 17 18 CoxFe3-xO4 19 FAmMNPs2 7.60 8.408 51 6.0 20.0* 41.0 29 249 20 /CoO 21 22 FAmMNPs3 10.0 8.4075 21 7.6 52.6* 66.5 147 300 Co0.82Fe2.18O4 23 24 25 *SQUID (4 T), **VSM (1 T) 26 27 28 Acetylacetonate precursors, Fe(acac)3 and Co(acac)2 are quite common in the 29 30 solvothermal synthesis of ferrite MNPs, however the role of Co(acac)3 as a precursor has 31 not been paid much attention. The use of the trivalent instead of the divalent cobalt 32 33 acetylacetonate precursor was examined in the hydrothermal synthesis of CoFe2O4 MNPs 34 35 in the presence of ODA26 and it led in variations of Co2+ amount in Td and Oh sites in the 36 37 spinel cell. Additionally, due to its almost simultaneous decomposition/dissociation rate 38 39 with the Fe (III) precursor, in contrast with the divalent cobalt precursor (slower 40 decomposition rate), it enhanced the nucleation process and resulted in different sizes as 41 42 shown before by us. 43 44 The Powder X-ray diffraction diagrams of the samples showed all the 45 46 characteristic peaks of the cubic spinel structure of CoFe2O4 (pdf card no. 22-1086) 47 (Figure 1). All samples were found to be pure phase cobalt ferrite except for sample 48 49 FAmMNPs2; the XRD diagram of this sample indicated an additional phase, that of the 50 51 cubic CoO (pdf card no. 42-1300). For the 2Fe(III):1Co(III) precursor ratio in the 52 53 ODA/DPE system (sample FAmMNPs2) it can be stated that part of the Co (III) 54 55 precursor was reduced by the amine (ODA) and formed a CoO phase through a side 56 57 58 59 12 60 ACS Paragon Plus Environment

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reaction, whereas for sample FAmMNPs3 the Co (II) precursor was predominantly consumed in the formation of the CoFe2O4 spinel cell. 80 533

70 440

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FAmMNPs3

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FAmMNPs2

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Figure 1. XRD diagrams of samples AmMNPs and FAmMNPs1-3; (▼ CoFe2O4 (221086), ▼CoO (42-1300)).

The average crystalline size of the samples was calculated by fitting the diffraction data with a pseudo-Voigt function (Jade6 Software) and it was found 10.0 nm for AmMNPs and 5.5, 7.6, 10.0 nm for samples FAmMNPs1-3 respectively. The lattice constants were calculated 8.420, 8.396, 8.408 and 8.4075 Å for samples AmMNPs, FAmMNPs1-3 respectively. The minor variation of lattice parameter towards larger values than the bulk's (8.3919 Å), suggests that the unit cell is under stress.27 EDS analysis showed that the composition of the samples varied from the generic formula of CoFe2O4 as it is shown in Table 1.



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Figure 2. TEM images of the AmMNPs and FAmMNPs1-3.

TEM imaging was used for the determination of the mean size, size distribution and morphology of the nanoparticles (Figure 2). The number weighted distributions were built by counting over 150 NPs for each sample and were fitted with a standard lognormal function. The mean particle size was 7.5 ± 0.4 nm for AmMNPs with narrow size distribution while the particles were well formed with distinct shape (tetragonal and spherical). The samples FAmMNPs1-3 consist of truncated particles of 3.0 ± 0.4, 6.0 ± 0.3 and 7.6 ± 0.3 nm respectively. The size differences were attributed to the nucleation and growth phenomena, which were defined by the metal precursors (dissociation process) and the adsorption and desorption of the surfactant on the nuclei surface in the absence and presence of the aprotic solvent accordingly28. 14 ACS Paragon Plus Environment

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The presence of the organic coating was certified by FT-IR spectroscopy and thermal analysis. The FT-IR spectra of the samples have substantially different features. In case of AmMNPs (Figure S2a) the presence of ODA and N-octadecylamide (N-ODA amide) on the MNPs is shown. The peak at ~1645 cm-1, was assigned to the carbonyl group of N-ODA amide,29 that was formed after the nucleophilic attack of –NH2 group to the C=O of the acetylacetonate ligand. Additionally, the strong N-H stretching at ~3303 cm-1 and the N-H wagging mode at ~724 cm-1 of ODA are present, while they are slightly downshifted compared to the same features of neat ODA (~3336 cm-1), due to the attachment of the organic molecule on the metal core.30 At ~2918, 2841 and 2931 cm-1 the asymmetric and symmetric stretching vibrations of the methylene groups, respectively, are observed.31-33 The FT-IR spectra of the samples FAmMNPs1-3 (Figure S2b), showed the characteristic vibrations of the acetylacetone ligand (1730, 1591, 1488 cm-1) in addition with ODA characteristic vibrations. The shoulder at ~2490 cm-1 and the low intensity asymmetric and symmetric stretching vibrations of the methylene groups at ~2918 cm-1 indicated that ODA was coordinated on the metal core through the NH2 group. The nonpolar solvent favored the formation of Hacac that was further bound on the MNPs according to the reaction pathway given before by us.14 The presence of –NH2 group on the MNPs surface was investigated further with the ninhydrin colorimetric assay. The absence or appearance of Ruhemann’s purple complex at λmax ~600 nm confirmed the formation of MNPs with amine-free (Figure S3a) and aminated surface (Figure S3b) respectively. The free -NH2 groups were found 0.0031 mg mL-1 for sample AmMNPs (0.02 mg mL-1 MNPs@DMF). Thermogravimetric data analysis of the samples was performed under nitrogen atmosphere (Figure S4). The weight loss by the decomposition of the organic coating was 36%, 46%, 51% and 21% for AmMNPs and FAmMNPs1-3 respectively. For the AmMNPs three main steps of weight loss were observed after 100 ˚C suggesting the existence of a bilayer (inner/outer layer) structure surrounding the NPs and/or different binding sites of the functional groups of ODA and N-ODA amide.34 The hydrocarbon chain decomposition occurred at 200∼450 ˚C, while the removal of the amine or amide group took place at higher temperature owing to the bonding with the metal core.14 The 15 ACS Paragon Plus Environment


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DTG analysis, showed a main loss for sample AmMNPs. For the amine–free MNPs (FAmMNPs1-3) the main mass reduction step begins at ~220 ˚C, whereas for the aminated MNPs at ~250 ˚C.

Magnetic Properties Moderate Ms values (Table 1) 45.0, 66.5 emu g-1 were found for AmMNPs and FAmMNPs3 respectively by SQUID measurements at a maximum field of 4 T (Figure S5a and S5d) and for sample FAmMNPs1 the Ms was found 44 emu g-1 by VSM measurement at a maximum field of 1 T (Figure S5b). The coercive field (Hc) values were 222, 127 and 147 Oe for AmMNPs, FAmMNPs1 and FAmMNPs3 respectively, which is significantly lower than the Hc value of the bulk material (5.4 kOe),35 and is attributed to the size (

Octadecylamine-Mediated Versatile Coating of CoFe2O4 NPs for the

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