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Magnetofluorescent Nanocomposite Comprised of Carboxymethyl Dextran Coated Superparamagnetic Iron Oxide Nanoparticles and β-Diketon Coordinated Europium Complexes Daewon Han 1 , Seung-Yun Han 2 , Nam Seob Lee 2 , Jongdae Shin 1,3 , Young Gil Jeong 2 , Hwan-Woo Park 1,3, * and Do Kyung Kim 2, * 1 2 3

*

Department of Cell Biology, Konyang University College of Medicine, Daejeon 302-718, Korea; [email protected] (D.H.); [email protected] (J.S.) Department of Anatomy, College of Medicine, Konyang University, Daejeon 302-718, Korea; [email protected] (S.-Y.H.); [email protected] (N.S.L.); [email protected] (Y.G.J.) Myunggok Medical Research Institute, Konyang University College of Medicine, Daejeon 302-718, Korea Correspondence: [email protected] (H.-W.P.); [email protected] (D.K.K.); Tel.: +82-42-600-8677 (H.-W.P.); +82-42-600-6445 (D.K.K.)

Received: 10 December 2018; Accepted: 29 December 2018; Published: 4 January 2019

Abstract: Red emitting europium (III) complexes Eu(TFAAN)3 (P(Oct)3 )3 (TFAAN = 2-(4,4,4-Trifluoroacetoacetyl)naphthalene, P(Oct)3 = trioctylphosphine) chelated on carboxymethyl dextran coated superparamagnetic iron oxide nanoparticles (CMD-SPIONs) was synthesized and the step wise synthetic process was reported. All the excitation spectra of distinctive photoluminesces were originated from f-f transition of EuIII with a strong red emission. The emission peaks are due to the hypersensitive transition 5 D0 →7 F2 at 621 nm and 5 D0 →7 F1 at 597 nm, 5 D0 →7 F0 at 584 nm. No significant change in PL properties due to addition of CMD-SPIONs was observed. The cytotoxic effects of different concentrations and incubation times of Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs were evaluated in HEK293T and HepG2 cells using the WST assay. The results imply that Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs are not affecting the cell viability without altering the apoptosis and necrosis in the range of 10 to 240 µg/mL concentrations. Keywords: europium complexes; superparamagnetic; iron oxide nanoparticles; magnetofluorescent

1. Introduction Non-invasive fluorescent [1] probes are required to evaluate the efficacy of the drug at the molecular level. The evaluation of the efficacy of the drug is also important from a biological point of view, as it traces the specific expression after administrating the drug into the cells [2]. Among these probes, fluorescence probes are excellent for tracking light emitted from the molecules after excitation at a specific wavelength because they have high sensitivity and spatial resolution [3]. One of the most important factor in selecting a bioimaging probe is the organic molecules/particles needed to be easily localize/internalize to particular organelles or sub-cellular sites. Currently, optical probes are classified into two categories; one is organic molecules such as fluorescein isothiocyanate (FITC) [4], Alexa Fluor [5], tetramethylrhodamine (TRITC) [6], cyanine (Cy3, Cy5, and Cy7), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and cyan fluorescent protein (CFP), etc., while the other consists of inorganic materials/quantum dots (QDs), such as CdSe/ZnS [7], InP/ZnS [8], CuInS/ZnS [9], and CH3 NH3 PbX3 (X = Cl, Br, I) [10], etc. QDs are very commonly used materials in biomedical applications such as in detection, biomarkers, and imaging agents. However, the ineffective uptake of QDs in living cells is an obstacle Nanomaterials 2019, 9, 62; doi:10.3390/nano9010062

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to its use in nanomedicine. A high dose of QDs is required to resolve the low localization in living cells. It is therefore significant to realize and strategy of designing an effective QDs for biomedical uses by understanding the permeability of cell membranes depending on the substances and functional groups present on the QD surface [11]. The synthesis of lanthanide complexes, which is a substance that emits light under ultraviolet light, has been extensively studied because of its broad range of applications. In recent years, the lanthanide complexes have been intensively investigated in nanomedicine because of their strong characteristics against anti-photobleaching compared with conventional fluorescent materials. They can be used in diagnostic kit, micro chemical detections, cell labeling, and other applications, because of their low cytotoxicity and high quantum yield [12]. At present, magnetic nanoparticles have been developed to improve the diagnosis and therapeutic effects of various diseases, and they are the most studied materials among nano-sized materials developed to date. Superparamagnetic iron oxide nanoparticles (SPION) have inherent beneficial characteristics, including magnetic properties, magnetized under external magnetic field, biocompatibility, high dispersivity after coating with biocompatible substance, cellular uptake, and a short blood half-life. These benefits are the most effective parameters for transporting drugs, proteins, and probes for nanomedicinal applications [13,14]. In this work, we try to develop a multi-purpose nanoprobe called magnetofluorescent [15,16] nanoparticles that can be traced noninvasively by MRI in-vivo and by confocal microscope in-vitro. SPIONs were prepared in the presence of dextran molecules and carboxymethyl group was activated with monochloroacetic acid followed by crosslinking the dextran (CLD) molecules with epichlorohydrin to cage-like structure tightly grafted on SPIONs. Finally, β-diketon coordinated europium complexes composed of 2-(4,4,4-trifluoroacetoacetyl)naphthalene (TFAAN), trioctylphophine (P(Oct)3 ), and EuIII were chelated on CLD-SPION. 2. Materials and Methods 2.1. Chemicals Ferric chloride hexahydrate (FeCl3 ·6H2 O), ferrous chloride tetrahydrate (FeCl2 ·4H2 O), ammonium hydroxide solution (28% in water), europium chloride hexahydrate (EuCl3 ·6H2 O), trioctylphophine (P(Oct)3 ), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and ethanol dialysis tubing cellulose membrane with a weight-cutoff (MWCO = 12,400) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Tokyo Chemical Industry Co. Ltd (Tokyo, Japan) supplied the 2-(4,4,4-Trifluoroacetoacetyl)naphthalene (TFAAN). Dextran T-10, epichlorohydrin and monochloroacetic acid (MCA) were supplied by DaeJung Chemicals and Metals Co. Ltd. (Kyunggi do, South Korea). 2.2. Characterization The particle size, morphology, and distribution quality of Eu(TFAAN)3 (P(Oct)3 )3 on CMD-SPION nanospheres were analyzed by Hitachi H-7650 TEM (EVISA, Tokyo, Japan). Fourier transform infrared (FT-IR) spectra were recorded using an ALPHA FT-IR Spectrometer equipped with Platinum ATR (Bruker, Billerica, MA, USA). Excitation and emission spectra were examined by fluorospectrophotometer (RF-5301PC Shimadzu, Kyoto, Japan) equipped with a 150 W xenon discharge lamp as a light source. Cells were observed using LSM 510 confocal laser scan microscope (Zessi, Inc., San Diego, CA, USA) and E800 epifluorescence microscopic (Nikon, Inc., Tokyo, Japan). 2.3. Synthesis of β-Diketon Coordinated Europium Complexes Europium complexes [17,18], Eu(TFAAN)3 (P(Oct)3 )3 , was prepared based on previously reported method with a minor modification. Europium chloride hexahydrate (EuCl3 ·6H2 O, 0.366 g, 1 mmol) was dissolved in 50 mL distilled H2 O. In similar way, 2-(4,4,4-Trifluoroacetoacetyl)naphthalene (TFAAN,

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0.266 g, 1 mmol) and trioctylphosphine (P(Oct) g, 1 and mmol) were dissolved(P(Oct) in 50 mL EtOH. Trifluoroacetoacetyl)naphthalene (TFAAN, 0.2663 ,g,0.370 1 mmol) trioctylphosphine 3, 0.370 g, 3+ , The final concentration of each stock solutions were 0.02 mmol. From the stock solution, 2 mL Eu 1 mmol) were dissolved in 50 mL EtOH. The final concentration of each stock solutions were 0.02 3+, 6 in 6 mL TFAAN, 6 mL P(Oct) 100 (28% water) NH46OH transferred touL 20 (28% mL glass bottle mmol. From the stock solution, mLµLEu mL TFAAN, mLwere P(Oct) 3, and 100 in water) 3 , and 2 with4OH a capped tightly and to the20 temperature was increased to 60 ◦ C in a water bathtemperature under magnetic NH were transferred mL glass bottle with a capped tightly and the was stirring. The product was precipitated by centrifuge, washed three times with distilled H O and increased to 60 °C in a water bath under magnetic stirring. The product was precipitated by 2 ◦ dried in vacuum oven at 70 C for 24 distilled h. The chemical structure Eu(TFAAN) (P(Oct) shown in centrifuge, washed three times with H2O and dried inofvacuum oven3 at 70 °C3 )for h. The 3 is 24 Scheme 1a. chemical structure of Eu(TFAAN)3(P(Oct)3)3 is shown in Scheme 1a.

Scheme 1.1. Schematic illustration of (a)offormation of europium complexes with 2-(4,4,4Schematic illustration (a) formation of europium complexes with 2-(4,4,4-Trifluoroacetoacetyl)naphthalene and trioctylphosphine (P(Oct) ), (b) in-situ Trifluoroacetoacetyl)naphthalene (TFAAN)(TFAAN) and trioctylphosphine (P(Oct)3), (b) in-situ synthesis of 3 synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) in dextran matrix, crosslinking the superparamagnetic iron oxide nanoparticles (SPIONs) in dextran matrix, crosslinking the dextran dextran molecules with epichlorohydrin and activation of carboxymethyl with monochloroacetic molecules with epichlorohydrin and activation of carboxymethyl group group with monochloroacetic acid; acid; (c) chelation of Eu(TFAAN) )3 on CMD-SPION. (c) chelation of Eu(TFAAN) 3(P(Oct) 3)3 on 3 CMD-SPION. 3 (P(Oct)

2.4. In situ Synthesis of Dextran Coated Superparamagnetic Iron Oxide Nanoparticles by Co-Precipitation 2.4. In situ Synthesis of Dextran Coated Superparamagnetic Iron Oxide Nanoparticles by Co-Precipitation Scheme 1b shows the overall procedure to prepare the CMD-SPIONs. N gas was directly flowed Scheme 1b shows the overall procedure to prepare the CMD-SPIONs. N22 gas was directly flowed into the solution for 30 min to remove the oxygen before the experiments. 5.46 g Iron(III) chloride into the solution for 30 min to remove the oxygen before the experiments. 5.46 g Iron(III) chloride hexahydrate and 1.99 g iron(II) chloride tetrahydrate were dissolved in 90 mL H2 O and 500 µL hexahydrate and 1.99 g iron(II) chloride tetrahydrate were dissolved in 90 mL H2O and 500 uL concentrated HCl by heating at 60 ◦ C in a water bath until all the salt was fully dissolved. After a concentrated HCl by heating at 60 °C in a water bath until all the salt was fully dissolved. After a transparent solution was achieved, the solution was topped up with H2 O to make a final volume of transparent solution was achieved, the solution was topped up with H2O to make a final volume of 100 mL to prepare the iron stock solution ([Fe2+ ] = 200 mM, [Fe3+ ] = 100 mM). After this, 1 g Dextran 100 mL to prepare the iron stock solution ([Fe2+] = 200 mM, [Fe3+] = 100 mM). After this, 1 g Dextran T-10 (Mw 10,000) was dissolved in 10 mL iron stock and add 90 mL H2 O. After the dextran was T-10 (Mw 10,000) was dissolved in 10 mL iron stock and add 90 mL H2O. After the dextran was fully fully dissolved, the solution was placed in an ice bath for 30 min. 10 mL concentrated ammonia dissolved, the solution was placed in an ice bath for 30 min. 10 mL concentrated ammonia was added was added dropwise under magnetic stirring and kept for 30 min. The temperature of the mixture dropwise under magnetic stirring and kept for 30 min. The temperature of the mixture was increased was increased to 70 ◦ C and kept for an additional 30 min. After the product was cooled to 25 ◦ C to 70 °C and kept for an additional 30 min. After the product was cooled to 25 °C (R. T.), the black (R. T.), the black solid was recovered by neodymium magnet and the supernatant was eliminated solid was recovered by neodymium magnet and the supernatant was eliminated by decantation. The by decantation. The solid precipitate was washed 5 times with H2 O to remove unreacted salts and solid precipitate was washed 5 times with H2O to remove unreacted salts and by-products. The final by-products. The final product, called SPIONs, was dispersed in 100 mL H2 O by probe sonicate product, called SPIONs, was dispersed in 100 mL H2O by probe sonicate for 1 min in an ice bath to for 1 min in an ice bath to form an extremely stable colloidal solution. Crosslinking between the form an extremely stable colloidal solution. Crosslinking between the dextran molecules surrounding dextran molecules surrounding on SPIONs was performed to attach the dextran more steadily by on SPIONs was performed to attach the dextran more steadily by following process; 10 mL 5 M NaOH was added in stock solution of SPIONs under vigorous magnetic stirring. 5 mL


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following process; 10 mL 5 M NaOH was added in stock solution of SPIONs under vigorous magnetic stirring. 5 mL epichlorohydrin was added into the mixture and kept for 24 h at 25 ◦ C under vigorous shaking by the shaker to avoid the phase separation between aqueous and organic layer. The constant shaking stimulates the chemical reaction between two different phases. After finalizing the reaction, the mixture solution was poured in dialysis tubing cellulose membrane with a weight-cutoff (MWCO = 12,400) and dialyzed against 5 L distilled H2 O. The distilled H2 O was changed with fresh one every 1 h for 5 times and left overnight. The conductivity of the H2 O was monitored by a conductivity meter to regulate the termination point of the washing progression. The solution was concentrated by vacuum dryer until a final volume of 20 mL and remarked as CLD-SPIONs. To activate the carboxylic group in dextran, carboxymethylation was accomplished though the following process; 10 mL 0.1 M NaOH was mixed with 10 mL CLD-SPIONs under magnetic stirring for 30 min at 25 ◦ C while directly purging N2 . After this, 0.443 MCA was added dropwise to the solution, and the temperature was increased to 60 ◦ C in an oil bath and kept for 60 min while N2 was flowing. After the reaction was completed, CMD-SPIONs was dialyzed against 5 L distilled H2 O to remove the impurities and kept at 4 ◦ C. 2.5. Magnetofluorescent Composite of Eu(TFAAN)3 (P(Oct)3 )3 @CMD-SPIONs 30 mg CMD-SPIONs in 10 mL deionized water was added in 1 mg Eu(TFAAN)3 (P(Oct)3 )3 in 10 mL EtOH and heated to 80 ◦ C in oil bath under magnetic stirring for 1 h. The sample was dialyzed against 5 L deionized water using a membrane tubing with a molecular cut-off 12,500 for three consecutive periods of 8 h. The final chemical structure of Eu(TFAAN)3 (P(Oct)3 )3 @CMD-SPIONs is shown in Scheme 1c. 2.6. Cell Culture Human embryonic kidney (HEK) 293T cells and human liver cancer cell line HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Welgene, Gyeongsangbuk-do, Korea) supplemented with 10% fetal bovine serum (FBS, Welgene, Korea) and 100 U/mL penicillin-streptomycin (Welgene, Korea). Culture conditions were maintained in a humidified atmosphere containing 5% CO2 at 37 ◦ C. 2.7. Immunofluorescence HEK293T cells or HepG2 cells were seeded onto each coverslip with 3 × 105 cells per coverslip in 24-well culture plates and then grown to 80% confluence. The cells were treated with indicated concentrations of Eu(TFAAN)3 (P(Oct)3 )3 @CMD-SPIONS dissolved in water for indicated time periods. Coverslips were washed once with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde. After washing, coverslips were mounted in 40 ,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) on glass slides. Samples were analyzed under an epifluorescence-equipped microscope (DM2500, Leica, Wetzlar, Germay). 2.8. Cytotoxicity Assay Cell viability was measured using WST-1 assay (Daeil Lab Service) according to the standard protocol of the manufacturer. Briefly, HEK293T cells and HepG2 cells were plated in 96-well plates at a concentration of 1 × 104 cells/well and treated with indicated concentrations of Eu(TFAAN)3 (P(Oct)3 )3 @CMD-SPIONS for indicated time periods. After incubated, 10 µL of WST-1 solution was added to each well and incubated for 30 min at 37 ◦ C under 5% CO2 incubator. After this, optical density of 96-well plates was measured in a microplate reader (Bio-Rad) at 450 nm and the absorbance values of the treated cells were expressed as a percentage of the absorbance values of the control.


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3. Results Nanomaterials 2019, 9, x FOR PEER REVIEW

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Figure 1 shows TEM images of carboxymethyl dextran coated superparamagnetic iron oxide Nanomaterials 2019, 9, x FOR PEER REVIEW 5 of 11 nanoparticles (CMD-SPIONs) and Eu(TFAAN) on CMD-SPIONs. The averageof particle diameter of SPIONs was around 12 nm with an irregular shape. After conjugation 3 (P(Oct) 3 )3 conjugated particle diameter of 3SPIONs SPIONs was around 12 nm nm with anirregular irregularshape. shape. After conjugation Eu(TFAAN) 3(P(Oct) )3, the was particles had some agglomeration. The After gray molecules are particle diameter of around 12 with an conjugation of of Eu(TFAAN) (P(Oct) , the particles had some agglomeration. The gray molecules are Eu(TFAAN) 3(P(Oct) 3)3,3 )as shown in Figure 1b and can be eliminated by washing with ethanol 3 3 Eu(TFAAN)3(P(Oct)3)3, the particles had some agglomeration. The gray molecules areby Eu(TFAAN) (P(Oct) Figure 1b and can bymagnet. washing with ethanol by centrifuge 3or applying an shown externalinmagnetic forces suchbe aseliminated neodymium 3 )3 , as Eu(TFAAN) 3(P(Oct)3)3, as shown in Figure 1b and can be eliminated by washing with ethanol by centrifuge centrifuge or or applying applying an an external external magnetic magnetic forces forces such such as as neodymium neodymium magnet. magnet.

Figure 1. TEM images of (a) carboxymethyl dextran coated superparamagnetic iron oxide nanoparticles (CMD-SPIONs) (b) Eu(TFAAN) 3(P(Oct)3)3 conjugated on CMD-SPIONs. Figure 1.1.TEM images of (a)ofcarboxymethyl dextran coated superparamagnetic iron oxide nanoparticles TEM images (a)and carboxymethyl dextran coated superparamagnetic iron oxide (CMD-SPIONs) and (b) Eu(TFAAN) CMD-SPIONs. nanoparticles (CMD-SPIONs) and (b) Eu(TFAAN) 3(P(Oct)3on )3 conjugated on CMD-SPIONs. 3 (P(Oct) 3 )3 conjugated

Differential scanning calorimetry (DSC) analysis was performed for CMD-SPION, Eu(TFAAN) 3(P(Oct) 3)3, and calorimetry Eu(TFAAN) chelated CMD-SPIONs (Figure DSC plot of Differential scanning (DSC) 3)3analysis was performed for 2). CMD-SPION, Differential scanning calorimetry 3(P(Oct) (DSC) analysis was performed for CMD-SPION, CMD-SPION exhibited the endothermic peaks at 75 °C and 164.6 °C and exothermic peak 136of °C, Eu(TFAAN) (P(Oct)33))3,3 ,and andEu(TFAAN) Eu(TFAAN)3(P(Oct) CMD-SPIONs(Figure (Figure2). 2). DSC DSCatplot plot 3 (P(Oct) 3 chelatedCMD-SPIONs Eu(TFAAN)33(P(Oct) 3)33)chelated of ◦ ◦ ◦ whereas Eu(TFAAN) 3)3 and Eu(TFAAN) 3(P(Oct) 3)3 164.6 chelated CMD SPIONs did notat CMD-SPION exhibited the peaks C and and C and and exothermic peak atappear 136 °C, C,at CMD-SPION exhibited3(P(Oct) the endothermic endothermic peaks at at 75 75 °C 164.6 °C exothermic peak 136 all. The temperature profile of Eu(TFAAN) 3 (P(Oct) 3 ) 3 system showed the distinctive peak at 54 °C whereas Eu(TFAAN) (P(Oct) ) and Eu(TFAAN) (P(Oct) ) chelated CMD SPIONs did not appear whereas Eu(TFAAN)33(P(Oct)33)33 and Eu(TFAAN)33(P(Oct)33)33chelated CMD SPIONs did not appear at atas ◦ it The appeared in DSCprofile curve of both Eu(TFAAN) 3(P(Oct) 3)3 andshowed Eu(TFAAN) 3(P(Oct)3)3peak chelated CMD all. The temperature profile of Eu(TFAAN) Eu(TFAAN) (P(Oct) the distinctive C 3 system all. temperature 33(P(Oct) 3)33)system showed the distinctive peak at at 54 54 °C as SPIONs. as it appeared in DSC curve of both Eu(TFAAN) (P(Oct) ) and Eu(TFAAN) (P(Oct) ) chelated 3 3 3 Eu(TFAAN)3(P(Oct) 3 3)3 chelated 3 3 it appeared in DSC curve of both Eu(TFAAN)3(P(Oct) 3)3 and CMD CMD SPIONs. SPIONs. 0.2 Eu(TFAAN)3(P(Oct)3)3

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Figure 2. Differential scanning calorimetry analysis of CMD-SPION and Eu(TFAAN)3 (P(Oct)3 )3 Figure 2. Differential scanning calorimetry analysis of CMD-SPION and Eu(TFAAN)3(P(Oct)3)3 chelated CMD-SPIONs. chelated Figure 2. CMD-SPIONs. Differential scanning calorimetry analysis of CMD-SPION and Eu(TFAAN)3(P(Oct)3)3 chelated CMD-SPIONs. Figure 3 shows PL spectra of (a) emission and (b) excitation profiles depending on different

Figure 3 shows PL spectra of (a) emission and (b) excitation profiles depending on different concentration of Eu(TFAAN)3 (P(Oct)3 )3 chelated on CMD-SPIONs. All the excitation spectra reveal a concentration of Eu(TFAAN) 3(P(Oct)3)3 chelated on CMD-SPIONs. All the excitation spectra reveal a Figure 3tendency shows PL spectra of (a) emission and (b) profiles depending different comparable of distinctive photoluminesces (PL)excitation coming from f-f transition of on EuIII with a III with a comparable tendency of distinctive photoluminesces (PL) coming from f-f transition of Eu concentration of Eu(TFAAN) 3)3 chelated on(EM) CMD-SPIONs. spectra reveal a strong red emission [19]. The3(P(Oct) maximum emission peak is dueAll to the the excitation hypersensitive transition strong red tendency emission [19]. The maximum emission (EM) is due to f-f thetransition hypersensitive comparable of distinctive photoluminesces (PL)peak coming from of EuIIItransition with a 5D0→7F2 at 621 nm and 5D0→7F1 at 597 nm, 5D0→7F0 at 584 nm. No significant change in PL properties strong red emission [19]. The maximum emission (EM) peak is due to the hypersensitive transition due to addition of CMD-SPIONs was observed. 5D 0→7F2 at 621 nm and 5D0→7F1 at 597 nm, 5D0→7F0 at 584 nm. No significant change in PL properties 4 shows zeta potentials of CMD-SPIONs and Eu(TFAAN)3(P(Oct)3)3 chelated on CMDdue toFigure addition of CMD-SPIONs was observed. SPIONs. Zeta (ζ) potential is used as a proper index for colloidal stability by evaluating the Figure 4 shows zeta potentials of CMD-SPIONs and Eu(TFAAN) 3(P(Oct)3)3 chelated on CMDquantification of the magnitude of the charge on the and the is generally SPIONs. Zeta (ζ) potential is used assurface a proper index for particles colloidal[20], stability byvalue evaluating the

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used to interpret and manipulate the stability of colloidal dispersion. The ζ-potentials of CMD5 D →7 F at 621 nm and 5 D →7 F at 597 nm, 5 D →7 F at 584 nm. No significant change in PL 0 is −17 2 0 13(P(Oct)3)3 chelated 0 0 CMD-SPIONs is changed to 9.7 mV. SPIONs mV and Eu(TFAAN) on properties due to addition of CMD-SPIONs was observed.

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600 600 used to interpret and manipulate the stability of colloidal dispersion. The ζ-potentials of CMD500 SPIONs is −17 mV and Eu(TFAAN)3(P(Oct)3)3 chelated on CMD-SPIONs is changed to 9.7 mV.

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400 Figure400 3. (a) Emission and (b) excitation spectra depending on different concentration of Figure 3. (a) Emission and (b) excitation spectra 300depending on different concentration of Eu(TFAAN)3 (P(Oct)3 )3 chelated on CMD-SPIONs. Emission spectra were measured at the excitation 200 200 Eu(TFAAN) 3(P(Oct) )3 chelated on CMD-SPIONs. wavelength of597360 3nm and excitation wavelength of Emission 621 nm. spectra were measured at the excitation 584 100

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wavelength of 360 nm and excitation wavelength of 621 nm. 0 0 575 600 625 650 675 700 330 360 390 420 450 Figure 4 shows zeta potentials of CMD-SPIONs 300 and Eu(TFAAN) 3 (P(Oct)3 )3 chelated on Wavelength (nm) Wavelength (nm) CMD-SPIONs. Zeta (ζ) potential is used as a proper index for colloidal stability by evaluating the (a) 50 quantification of magnitude of the surface spectra charge on the particles [20], andconcentration the value is generally Figure 3. (a) the Emission and (b) excitation depending on different of used and manipulate theCMD-SPIONs. stability of colloidal The ζ-potentials CMD-SPIONs 40 to interpret Eu(TFAAN) 3(P(Oct) 3)3 chelated on Emissiondispersion. spectra were measured at theofexcitation is −wavelength 17 mV andofEu(TFAAN) (P(Oct)3 )wavelength CMD-SPIONs is changed to 9.7 mV. 360 nm and 3excitation of 621 nm. 3 chelated on 657

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10 Figure 4. (a) Zeta Potentials of CMD-SPION and Eu(TFAAN)3(P(Oct)3)3 chelated on CMD-SPIONs. Photograph images of CMD-SPION (left) and Eu(TFAAN)3(P(Oct)3)3 chelated on CMD-SPIONs (right) 0 -200 -150 -100 -50 0 50 100 150 200 Zeta Potential (mV) (b) daylight, (c) daylight + UV light at 365 nm, and (d) UV light at 365 nm. dispersed in water under

Figure 4. (a) Zeta Potentials of CMD-SPION and Eu(TFAAN)3 (P(Oct)3 )3 chelated on CMD-SPIONs.

Figure 4. (a) Zeta Potentials of CMD-SPION and Eu(TFAAN)3(P(Oct)3)3 chelated on CMD-SPIONs. Figure 5 shows FTIR spectra of Eu(TFAAN) 3(P(Oct)3)3, CMD-SPIONs and Eu(TFAAN)3(P(Oct)3)3 Photographimages imagesofofCMD-SPION CMD-SPION(left) (left)and andEu(TFAAN) Eu(TFAAN)3(P(Oct) )3 chelatedononCMD-SPIONs CMD-SPIONs(right) (right) 3 (P(Oct) Photograph 3)33 chelated −1 coming from OH-stretching chelateddispersed on CMD-SPIONs show absorption peaks around 3300 cm in water under (b) daylight, (c) daylight + UV light at 365 nm, and (d) UV light at 365 nm. dispersed in water under (b) daylight, (c) daylight + UV light at 365 nm, and (d) UV light at 365 nm. vibrations of water molecules and 2922 cm−1 can be allocated to the sp3 bonding of C–H. In the CMD Figure 5 shows FTIR spectra of Eu(TFAAN) and Eu(TFAAN) 3 (P(Oct) 3 )3 , CMD-SPIONs 3 (P(Oct)3 )3 spectrum, deformation vibration which appears at 1250 cm−1 and ν(C–O) vibration Figure 5 shows FTIR spectra ofδ(C–OH) Eu(TFAAN) 3(P(Oct) 3)3, CMD-SPIONs and Eu(TFAAN) 3(P(Oct)3)3around −1 coming from OH-stretching chelated on CMD-SPIONs show absorption peaks around 3300 cm −1 −1 −1 1150 cm . on TheCMD-SPIONs absorption at 1462absorption cm is attributed to C=C 1688from cm OH-stretching is assigned to C=O chelated show peaks around 3300bond cm−1and coming 3 bonding of C–H. In the CMD vibrations of water molecules and 2922 cm−1−1 can be allocated to the sp 3 −1 vibrations of water molecules 2922of cmP=O can be allocated to theare spappeared bonding of In the CMD bond. The stretching vibrationand bands (1061 cm ) bond inC–H. Eu(TFAAN) 3(P(Oct)3)3 spectrum, deformation vibration δ(C–OH) which appears at 1250 cm−1−1 and ν(C–O) vibration around spectrum, deformation vibration δ(C–OH) which appears at 1250 cm and ν(C–O) vibration around and Eu(TFAAN) 3)3 chelated on−CMD-SPIONs. results imply that Eu(TFAAN)3(P(Oct)3)3 −1 . The3(P(Oct) 1 is attributed to These 1150 cm absorption at 1462 cm C=C bond and 1688 cm−1−1 is assigned to C=O 1150 cm−1. The absorption at magnetic 1462 cm−1 nanoparticles. is attributed to−C=C bond and 1688 cm is assigned to C=O was successively grafted on bond. The stretching vibration bands of P=O (1061 cm 1 ) bond are appeared in Eu(TFAAN) (P(Oct) ) 3 3 3 bond. The stretching vibration bands of P=O (1061 cm−1) bond are appeared in Eu(TFAAN)3(P(Oct) 3)3 and Eu(TFAAN)3 (P(Oct)3 )3 chelated on CMD-SPIONs. These results imply that Eu(TFAAN)3 (P(Oct)3 )3 and Eu(TFAAN)3(P(Oct)3)3 chelated on CMD-SPIONs. These results imply that Eu(TFAAN)3(P(Oct)3)3 was successively grafted on magnetic nanoparticles. was successively grafted on magnetic nanoparticles. The cytotoxic effects of concentrations and incubation times of Eu(TFAAN)3 (P(Oct)3 )3 chelated Eu(TFAAN) (P(Oct) ) @CMD-SPION CMD-SPIONs were evaluated in HEK293T and HepG2 cells using the WST assay. (Figure 6) Figure 6a shows the cytotoxic effects of HEK293T and HepG2 cells treated with the indicated concentration CMD-SPION Eu(TFAAN) (P(Oct) ) @CMD-SPION of Eu(TFAAN)3 (P(Oct)3 )3 @CMD-SPIONs for 6 h. Cell viability was measured by WST-1 assay. Data are shown as mean ± s.e.m. (n = 3). Figure 6b shows cytotoxic effects of HEK293T and Eu(TFAAN) (P(Oct) ) CMD-SPION HepG2 cells treated with 72 µg/mL Eu(TFAAN)3 (P(Oct)3 )3 @CMD-SPIONs for indicated periods of time. Cell viability was measured by WST-1 assay. Data are shown as mean ± s.e.m. (n = 3) % Transmittance

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Figure 5 shows FTIR spectra of Eu(TFAAN)3(P(Oct)3)3, CMD-SPIONs and Eu(TFAAN)3(P(Oct)3)3 chelated on CMD-SPIONs show absorption peaks around 3300 cm−1 coming from OH-stretching vibrations of water molecules and 2922 cm−1 can be allocated to the sp3 bonding of C–H. In the CMD spectrum, deformation vibration δ(C–OH) which appears at 1250 cm−1 and ν(C–O) vibration around Nanomaterials 2019, 9, 62 7 of 11 1150 cm−1. The absorption at 1462 cm−1 is attributed to C=C bond and 1688 cm−1 is assigned to C=O −1 bond. The stretching vibration bands of P=O (1061 cm ) bond are appeared in Eu(TFAAN)3(P(Oct)3)3 andFigure Eu(TFAAN) 3(P(Oct) 3)3 chelated on CMD-SPIONs. results imply that Eu(TFAAN) 3(P(Oct)3)3 6c,d shows the results of HEK293T (c) and HepG2These cells (d) treated the indicated concentration wasofsuccessively grafted3 )on magnetic nanoparticles. Eu(TFAAN)3 (P(Oct) @CMD-SPIONs for 24 h. The cells were stained with Muse Annexin V and 3 Dead Cell reagent and then analyzed for apoptosis by the Muse Cell Analyzer.

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Figure 5. FT-IR spectraofofEu(TFAAN) Eu(TFAAN)33(P(Oct) (P(Oct)33))33,, CMD-SPION CMD-SPION and 3 (P(Oct) 3 )3 chelated Figure 5. FT-IR spectra andEu(TFAAN) Eu(TFAAN) 3(P(Oct) 3)3 chelated on 7 of 11 CMD-SPIONs.

Nanomaterials 2019, 9, x FOR PEER REVIEW on CMD-SPIONs.

Figure6. Cellular 6. Cellular cytotoxicity of Eu(TFAAN) (P(Oct)3 )3 @CMD-SPIONs and Figure cytotoxicity of Eu(TFAAN) 3(P(Oct)3)33@CMD-SPIONs in HEK293Tin andHEK293T HepG2 cells. HepG2 cells. (a) HepG2 were treated indicated concentrationofof (a) HEK293T andHEK293T HepG2 and cells werecellstreated with with the the indicated concentration Eu(TFAAN)3(P(Oct) )3 @CMD-SPIONs Dataare areshown shownasasmean mean±±s.e.m. s.e.m.(n(n= =3)3)(b) (b)HEK293T HEK293T Eu(TFAAN) 3)33@CMD-SPIONs forfor6 6h.h.Data 3 (P(Oct) andHepG2 HepG2cells cellswere were treated treated with with 72 µg/mL (P(Oct)33)3)@CMD-SPIONs @CMD-SPIONs for indicated and μg/mL Eu(TFAAN)33(P(Oct) for indicated 3 periods of time. Data are shown as mean ± s.e.m. (n = 3) (c,d) HEK293T (c) and HepG2 cells (d)were were periods of time. Data are shown as mean ± s.e.m. (n = 3) (c, d) HEK293T (c) and HepG2 cells (d) treated the indicated concentration of Eu(TFAAN) (P(Oct) ) @CMD-SPIONs for 24 h. treated the indicated concentration of Eu(TFAAN)3(P(Oct) 3)33@CMD-SPIONs for 24 h. 3 3

Figure 7a,b shows intracellular uptake and of Eu(TFAAN) 3 (P(Oct) 3 )3 @CMD-SPIONs The cytotoxic effects of concentrations and distribution incubation times of Eu(TFAAN) 3(P(Oct) 3)3 chelated in HEK293T and HepG2 cells. in Representative images of cells HEK293T HepG2 cells (b) treated with CMD-SPIONs were evaluated HEK293T and HepG2 using(a) theand WST assay. (Figure 6) Figure

6a shows the cytotoxic effects of HEK293T and HepG2 cells treated with the indicated concentration of Eu(TFAAN)3(P(Oct)3)3@CMD-SPIONs for 6 h. Cell viability was measured by WST-1 assay. Data are shown as mean ± s.e.m. (n = 3). Figure 6b shows cytotoxic effects of HEK293T and HepG2 cells treated with 72 μg/ml Eu(TFAAN)3(P(Oct)3)3@CMD-SPIONs for indicated periods of time. Cell viability was measured by WST-1 assay. Data are shown as mean ± s.e.m. (n = 3) Figure 6c,d shows


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vehicle (Control) or Eu(TFAAN)3 (P(Oct)3 )3 @CMD-SPIONs at 72 µg/mL concentration for 4 h. Nuclei were stained with (blue). Scale bar, 20 µm. Nanomaterials 2019, 9, xDAPI FOR PEER REVIEW 8 of 11

Figure 7. 7. Intracellular Intracellularuptake uptake images of HEK293T (a) HepG2 and HepG2 cells (b) with treated with(Control) vehicle images of HEK293T (a) and cells (b) treated vehicle (Control) or Eu(TFAAN) 3 (P(Oct) 3 ) 3 @CMD-SPIONs. Nuclei were stained with DAPI (blue). Scale bar, or Eu(TFAAN) (P(Oct) ) @CMD-SPIONs. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. 3 3 3 20 µ m.

4. Discussion

4. Discussion The crosslinking reaction of dextran [21] by epichlorohydrin happen in inter- or intra-molecular formsThe and are graftedreaction on SPIONs surfaces. The moleculeshappen are transformed the surface crosslinking of dextran [21] bydextran epichlorohydrin in inter- orfrom intra-molecular of SPIONs into a strong and rigid structure, resulting in a heterogeneous solid structure on the forms and are grafted on SPIONs surfaces. The dextran molecules are transformed from the surface SPIONs surface. core-shell structure maintains the intermolecular bondingsolid of physically of SPIONs into aThe strong and rigid structure, resulting in a heterogeneous structurebonded on the intermolecular materials and prevents separation in the aqueous solution. The epoxy moiety of SPIONs surface. The core-shell structure maintains the intermolecular bonding of physically bonded epichlorohydrin alkylates OH groups and its epoxy group interacts with other OH groups of the intermolecular materials and prevents separation in the aqueous solution. The epoxy moiety of dextran to form the corresponding interanditsintramolecular epichlorohydrin alkylates OH groups and epoxy group crosslinks. interacts with other OH groups of the In general, the magnetic nanoparticles have remanence magnetization dextran to form the corresponding inter- and intramolecular crosslinks. even though it is classified as superparamagnetic material. nanoparticles The residual magnetic forces will induce the interactions between In general, the magnetic have remanence magnetization even though it is the magnetic particles resulting in large coagulation in the diameter range of 50–300 nm. To avoid classified as superparamagnetic material. The residual magnetic forces will induce the interactions the agglomeration, nonmagnetic substances were grafted oninthe of range SPIONs. In thisnm. study, between the magnetic particles resulting in large coagulation thesurface diameter of 50–300 To in situ formation of SPIONs was introduced to resolve the difficulties caused by coagulation during avoid the agglomeration, nonmagnetic substances were grafted on the surface of SPIONs. In this the synthesis magneticofnanoparticles. Dextran molecules in the the difficulties water formcaused polymeric matrix and study, in situ of formation SPIONs was introduced to resolve by coagulation 2+ and Fe3+ ions are located in dextran molecules. In this manner, SPIONs nucleate inside the Fe during the synthesis of magnetic nanoparticles. Dextran molecules in the water form polymeric matrix and and Fe retain the among particlesmolecules. while the In particles are grown by thermal 2+ and 3+ ions are matrix Fedistance locatedthe in dextran this manner, SPIONs nucleateenergy. inside The inter-particular forces due to the dipole-dipole interaction are adequate to elude the of the matrix and retain the distance among the particles while the particles are grown stimulus by thermal residual The magnetization resulting in forming constant colloidalare suspension. benefits energy. inter-particular forces due to exceptionally the dipole-dipole interaction adequateMore to elude the of using carbohydrate (i.e., dextran) are that it is biocompatible along with versatile derivatives by stimulus of residual magnetization resulting in forming exceptionally constant colloidal suspension. ◦ C was disappeared because TFAAN activation of specific functional groups. The DSC peak at 54 More benefits of using carbohydrate (i.e., dextran) are that it is biocompatible along with versatile and P(Oct)3 by molecules were each other resulting in disappearing endothermic and derivatives activation of diffused specific into functional groups. The DSC peak at 54 the °C was disappeared exothermic peaks, indicating that each molecule was changed into amorphous phase. In addition, because TFAAN and P(Oct)3 molecules were diffused into each other resulting in disappearing the

endothermic and exothermic peaks, indicating that each molecule was changed into amorphous phase. In addition, the mass of Eu(TFAAN)3(P(Oct)3)3 is relatively larger than that of CMD-SPIONs when they are grafted and is not observed even with exothermic or endothermic peaks. Approximately 20% of EM quenching was observed by comparing the EM intensity of Eu(TFAAN)3(P(Oct)3)3 chelated on CMD-SPIONs and Eu(TFAAN)3(P(Oct)3)3 at 621 nm. As the


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the mass of Eu(TFAAN)3 (P(Oct)3 )3 is relatively larger than that of CMD-SPIONs when they are grafted and is not observed even with exothermic or endothermic peaks. Approximately 20% of EM quenching was observed by comparing the EM intensity of Eu(TFAAN)3 (P(Oct)3 )3 chelated on CMD-SPIONs and Eu(TFAAN)3 (P(Oct)3 )3 at 621 nm. As the concentration of Eu(TFAAN)3 (P(Oct)3 )3 against CMD-SPIONs was increased, 5 D0 →7 F2 was proportionally amplified with a virtually Gaussian shape. The PL of Eu(TFAAN)3 (P(Oct)3 )3 and Eu(TFAAN)3 (P(Oct)3 )3 @CMD-SPIONS is analyzed by PL intensity ratio of 5 D0 →7 F2 and 5 D0 →7 F1 . 5 D0 →7 F1 is comparatively strong [22], which corresponds to the magnetic dipole transition, has an independent intensity value inherently not affected by coordination environment. Therefore, the intensity values of the 5 D0 →7 F1 transition that are forbidden both for magnetic and electric dipole and can be used for comparison. In opposition, the intensity value of 5 D0 →7 F2 is an electric dipole transition affected by the physicochemical change values around the EuIII . ζ-potentials of CMD-SPIONs have a rather lower value but are extremely stable in water because CMD on SPIONs forms hyper branched matrix in water resulting in localization of SPIONs inside the net-like matrix. For this reason, CMD-SPIONs are exceptionally consistent without coagulation/flocculation even though the value of ζ-potentials is relatively low. The net electro charge on CMD-SPION shows opposite values after modifying the surface with Eu(TFAAN)3 (P(Oct)3 )3 . Figure 4b–d shows digital images of CMD-SPION (left) and Eu(TFAAN)3 (P(Oct)3 )3 chelated on CMD-SPIONs (right) dispersed in water under (b) daylight, (c) daylight + UV light at 365 nm, and (d) UV light at 365 nm. Eu(TFAAN)3 (P(Oct)3 )3 chelated on CMD-SPIONs exhibited quite bright luminesce with a red color under excitation with a UV light at 365 nm [23]. The cytotoxicity of 6 h after treatment with a different concentration of Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs (10 to 240 µg/mL) show that the cell viability in the range of 10 to 240 µg/mL concentrations was more than 95%. Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs were not toxic to HEK293T and HepG2 cells even at a concentration of 240 µg/mL. At all time periods, no significant difference in cell viability was observed for all two cell types above when treated with Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs (Figure 6b). In addition, we determined the apoptotic effect of Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs in HEK293T and HepG2 cells by using Annexin V and dead cell reagent labeling flow cytometry. The four-quadrant plots in each panel show the necrotic cells (upper left), the late apoptotic cells (upper right), the viable cells (lower left), and the early apoptotic cells (lower right). Both cells treated with Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs revealed four-quadrant plots similar to those of the vehicle-treated cells (Figure 6c,d). These results indicate that Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs do not affect cell viability and do not alter the cell death program [24]. The intracellular uptake of Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs was evaluated in HEK293T and HepG2 cells using an epifluorescence-equipped microscopy. Internalization of Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs by HEK293T and HepG2 cells was not observed after 1 h incubation. After 6 h incubation, the cellular uptake of Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs in both cells occurred and was distributed within the cytoplasm (Figure 7). Since the europium complexes synthesized in this study have emission spectra at 619 nm when excited in the UV range, Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs do not require an additional red tracker. 5. Conclusions We designed and synthesized a novel magnetofluorescent nanoprobe comprising of red emitting europium (III) complexes Eu(TFAAN)3 (P(Oct)3 )3 chelated on carboxymethyl dextran coated CMD-SPIONs that can be traced noninvasively by MRI in-vivo and by confocal microscope in-vitro. All the excitation spectra distinctive photoluminesces came from f-f transition of EuIII with a strong red emission. No significant change in PL properties due to addition of CMD-SPIONs was observed. The cell viability measured in HEK293T and HepG2 cells shows that Eu(TFAAN)3 (P(Oct)3 )3 chelated


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CMD-SPIONs do not affect cell viability and do not alter apoptosis and necrosis in the range of 10 to 240 µg/mL concentrations. At 4 h after incubation, the red fluorescence from Eu(TFAAN)3 (P(Oct)3 )3 chelated CMD-SPIONs was mainly located in the cytoplasm with no significant cytotoxicity. Author Contributions: H.-W.P. and D.K.K. designed the experiments and wrote the paper; D.H. performed the experiments and wrote the paper; S.-Y.H., N.S.L. and J.S. analyzed the data; Y.G.J. discussed the results. Funding: This research was supported by grants from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (No. NRF-2017R1A2B4005167) and the Ministry of Education (No. NRF-2017R1A6A1A03015713). This research was also supported by a grant of the Korean Health Technology R&D Project funded by the Ministry of Health & Welfare, Republic of Korea (No. HI17C1238). Conflicts of Interest: The authors declare no conflict of interest.

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