Accepted Manuscript

Title: Synthesis and Characterization of Bodipy

Functionalized Magnetic Iron oxide Nanoparticles for
Potential Bioimaging Applications

Author: Seda Demirel Topel Onder

Topel R. Beklem
Bostancoglu A. Tansu Koparal
PII:
DOI:
Reference:

S0927-7765(15)00059-4
http://dx.doi.org/doi:10.1016/j.colsurfb.2015.01.043
COLSUB 6876

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date:
Revised date:
Accepted date:

30-11-2014
9-1-2015
27-1-2015

Topel, R.B. Bostancioglu, A.T. Koparal,

Please cite this article as: S.D. Topel, O.
Synthesis and Characterization of Bodipy Functionalized Magnetic Iron oxide
Nanoparticles for Potential Bioimaging Applications, Colloids and Surfaces B:
Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.01.043
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Graphical Abstract

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Highlights

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Multifunctional magnetic nanoparticles using a Bodipy fluorophore (BOD-MNPs) were synthesized

around 10 nm diameter.

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The results from in vitro tests are promising for the bio-imaging of cancer cells.

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The BOD-MNPs can easily penetrate into the cell cytoplasm in the living cells with their own

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fluorescence properties.

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The BOD-MNPs have no cytotoxic effect to A549 and Ishikawa cells.

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14

Synthesis and Characterization of Bodipy Functionalized Magnetic

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Iron oxide Nanoparticles for Potential Bioimaging Applications

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Seda Demirel Topela*, nder Topela*, R. Beklem Bostancolub, A. Tansu Koparalb

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Department of Chemistry, Faculty of Science, Akdeniz University, 07058, Antalya, Turkey

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Department of Biology, Faculty of Science, Anadolu University, Eskiehir, Turkey

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ABSTRACT

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Multifunctional magnetic nanoparticles were synthesized for potential bio-imaging applications.

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Uniform PEI coated magnetic Fe3O4 (PEI-Fe3O4) nanoparticles were prepared by a modified co-

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precipitation method and then covalently conjugated with a fluorophore molecule, Bodipy-5 by

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the DCC/DMAP coupling reaction. The covalent binding of Bodipy-5 to the PEI coated magnetic

29

Fe3O4 nanoparticles were confirmed by means of FTIR and XPS measurements. The imaging

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ability of the Bodipy coated magnetic nanoparticles was determined on two human cancer cells,

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A549 (human lung adenocarcinoma epithelial) and Ishikawa (endometrial adenocarcinoma), for

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the first time. Cytotoxicity of BOD-MNPs was evaluated in both cancer cells and healthy human

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umbilicial vein endothelial cell line (HUVEC) by standard MTT (3-(4,5-dimethythiazol-2-yl)-

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2,5-diphenyl tetrazolium bromide) assay. In vitro activities of the nanoparticles were also

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investigated.

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Key words: Magnetic nanoparticles, Bodipy, bio-imaging, cytotoxicity, multifunctional

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nanoparticles

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Corresponding authors: sedademirel@akdeniz.edu.tr, ondertopel@akdeniz.edu.tr

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1. Introduction

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In the last decade, magnetic nanostructures have become a focus of many researches at

45

chemistry-biology interface due to their biomedical applications such as magnetic bio-separation

46

[1], magnetically targeted drug delivery[2], hyperthermia [3], magnetic resonance imaging [4],

47

magnetofection [5] etc. These kind of nanostructures can be easily designed based on metallic,

48

bimetallic, and iron oxide nanoparticles possessing magnetic property [6,7]. Especially the

49

superparamagnetic iron oxide nanoparticles (SPIONs) have been widely applied to many area

50

due to their inoffensive toxicity profile [8,9] and easily functionalizing property with small

51

molecules and other nanostructures, which may open up many application possibilities. The

52

SPIONs may also be functionalized with drugs, fluorescent dye molecules, various hydrophilic

53

and hydrophobic coating agents such as poly(ethyleneglycol) (PEG) [10], dextran [11],

54

polyvinylpyrrolidone (PVP) [12], polyethylenimine (PEI) [13] fatty acids [14], polyvinyl alcohol

55

(PVA) [15], polyacrylic acid [16]. Among the functionalizing entities, it is well-known that

56

especially fluorescent dyes such as DAPI Hoescht, Mitotracker, Alexa Fluor, Allophycocyanin

57

are commonly used for biological labelling and staining. Nowadays multifunctional nanoparticles

58

having magnetic and fluorescence properties are of considerable interest. Recent works on the

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magnetic-fluorescent nanostructures are promising for potential medical applications of such as

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imaging, bio-and chemo-sensing, drug delivery and therapy systems [17]. However, the problems

61

such as the quenching of the fluorophor, the depletion of magnetization and the surface chemistry

62

to cellular uptake are required to achieve for the further applications. Several synthesis strategies

63

have been reported to overcome the challenges. For example, Gun`ko and co-workers prepared

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magnetic-fluoresent nanocomposites including porphyrin dye on the surface of the magnetic

65

nanoparticles [18]. The fabrication of these nanocomposites was accomplished by covalent

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bonding of porphyrin to magnetic nanoparticles via an appropriate spacer. They observed that the

67

resultant particles maintained their fluorescent and magnetic properties.

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Recently, a new class of fluorescent dye molecules, i.e. the boron-dipyrromethene (Bodipy)

70

dyes, have been received a considerable attention. These classes of fluorescent molecules exhibit

71

a small Stokes shift, high fluorescence quantum yields, sharp excitation-emission peaks and good

72

photochemical stability. Due to their unique photophysical properties, Bodipy dyes are of

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considerable interest for potential applications such as bioimaging, sensor applications [19]. Jung

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and co-workers [20] reported a Pb+2 sensor based on Bodipy receptor attached covalently to the

75

surface of Fe3O4@SiO2 core/shell nanoparticles. In this report, Fe3O4@SiO2 nanoparticles were

76

prepared by encapsulation of Fe3O4 nanocrystals within silica shells via microemulsion method.

77

Then, Bodipy fluorophore having triethoxysilane functional groups was covalently attached on

78

the surface of Fe3O4@SiO2 nanoparticles by sol-gel method. Reiser and co-workers [21] reported

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noncovalent functionalization of pyrene-tagged Bodipy fluorescent dye through - interactions

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on the surface of the carbon-coated cobalt nanoparticles. They functionalized covalently Bodipy

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dye on the carbon-coated cobalt nanoparticles via click chemistry. Their photophysical

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measurements on the Bodipy conjugated nanostructures via covalent bonding and noncovalent

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immobilization exhibit strong fluorescence property.

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The present study describes preparation of multifunctional magnetic nanocomposites

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containing a fluorescent Bodipy dye, and a detailed investigation of their bio-imaging and bio-

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labelling ability on the living cells. The synthesis of new Bodipy-magnetic iron oxide

88

nanocomposites was achieved by conjugating a Bodipy derivative having carboxylic acid

89

functional group on the PEI coated iron oxide nanoparticles by means of the DCC/DMAP

90

coupling reaction. The structural, morphological and optical properties of these nanocomposites

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as well as bio-imaging ability were discussed in detail. The imaging ability of the Bodipy

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conjugated magnetic nanocomposites (BOD-MNPs) was evaluated on two human cancer cells,

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A549 (human lung adenocarcinoma epithelial) and Ishikawa (endometrial adenocarcinoma), by

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performing fluorescence microscopy measurements while cytotoxic activity of the BOD-MNPs

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was tested on the healthy human umbilicial vein endothelial cell line (HUVEC) as well as the

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A549 and Ishikawa cells by standard MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium

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bromide) assay. Fluoresence microscopy upon ultraviolet (UV) excitation was achieved to access

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the potentialities of these Bodipy conjugated magnetic nanocomposites for biolabelling

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applications.

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2. Materials and methods

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2.1. Materials

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p-hydroxybenzaldehyde,

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6-bromohexanoic

acid,

benzo-18-crown-6,

3-ethyl-2,4-

dimethylpyrolle, trifluoroacetic acid (TFA), p-chloranil, triethylamine (TEA), BF3.OEt2,

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iron(III)chloride hexahydrate (FeCl3.6H2O) and iron(II)chloride tetrahydrate (FeCl2.4H2O),

107

polyethyleneimine (PEI), sodium hydroxide (NaOH) and other solvents (Merck) were used as

108

purchased. Purification of the BODIPY dyes was performed by flash column chromatography

109

using thickwalled glass columns and "flash grade" silica gel (Merck 230400 mesh). Purity of

110

the compounds was monitored by TLC on silica gel 60 F254s, aluminium sheets plates (Merck).

111

All commercially available reagents and reactants were obtained in reagent grade and used

112

without purification.

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2.2. Synthesis of 2,6-diethyl-1,3,5,7-tetramethyl-8-(4-(5-carboxypentyloxi)phenyl-4,4-difloro-4-

115

bora-3a,4a-diaza-s-indacen (5)

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The

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synthesis

of

Bodipy-5

[2,6-diethyl-1,3,5,7-tetramethyl-8-(4-(5-carboxy

pentyloxi)phenyl-4,4-difloro-4-bora-3a,4a-diaza-s-indacen] are summarized in Scheme S1. The

119

compound 3 ( 6-(4-formylphenoxy)hexanoic acid) was synthesized at first step as following: p-

120

hydoxybenzaldehyde (1 g, 8.18 mmol) was dissolved in dry acetonitrile (20 mL). K2CO3 (6.8 g,

121

49.08 mmol) and benzo-18-crown-6 (25 mg, 0.28 mmol) were added to the first solution. Into

122

this mixture, 6-bromohexanoic acid (2.4 g, 12.28 mmol) which dissolved in dry acetonitrile (15

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mL) was added. The reaction mixture was refluxed for overnight. The solid part of the reaction

124

mixture was filtered off and washed for two times with cold acetonitrile (15 mL), then dissolved

125

in water (15mL) and neutralized by 4 M HCl. White precipitates were filtered and dried. As a

126

result compound, 6-(4-formylphenoxy)hexanoic acid (2.45 g) was obtained in 58 % yield. 1H-

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NMR (400 MHz, CDCl3): ppm; 9.49 (s,1H), 7.51 (d, 2H, J=8.4 Hz), 6.70 (d, 2H, J=8.4 Hz),

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3.75 (t, 2H, J=6.3 Hz), 2.01 (t, 2H, J=7.2 Hz), 1.56-1.49 (m, 2H), 1.41-1.34 (m, 2H), 1.25-1.19

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(m, 2H). 13C-NMR (100 MHz, CDCl3): ppm; 195.1(C=O), 176.5 (C=O), 166.0 (Ar-Cipso), 136.0

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(Ar-C), 130 (Ar-Cipso), 118.7 (Ar-C), 72.0 (CH2), 37.8 (CH2), 32.5 (CH2), 29.4 (CH2), 28.4

131

(CH2).

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Bodipy-5 (5) was synthesized by reacting the compound 3 with 3-ethyl-2,4-dimethylpyrolle

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(4). In an oven dried 250 mL round bottom flask, dichloromethane (DCM) (200 mL) was added

135

and bubbled under nitrogen gas for 20 min. 6-(4-formylphenoxy)hexanoic acid (3) (700 mg, 2.96

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mmol) was dissolved in this flask. Then, 3-ethyl-2,4-dimethylpyrolle (4) (729.3 mg, 5.92 mmol)

137

and trifluoroaceticacid (TFA) (three drops) were added and stirred at room temperature for

138

overnight. p-chloranil (800 mg, 3.26 mmol) was added into this reaction mixture and stirred at

139

room temperature for 5 hours. After that, triethylamine (TEA) (5 mL, 35.82 mmol) and BF3.OEt2

140

(5 mL) were added. The reaction mixture was stirred further 30 min, then extracted with the brine

141

solution (3x100 mL). Organic phase was dried over Na2SO4 then filtered and evaporated. The

142

final mixture was purified by column chromatography using CHCl3-MeOH (3%) eluent system.
1

H-NMR (400 MHz, CDCl3): ppm; 7.16 (d, 2H, J=8.6 Hz, Ar-CH), 7.00 (d, 2H, J=8.6 Hz, Ar-

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CH), 4.03 (t, 2H, J=6.4 Hz, CH2), 2.56 (s, 6H, CH3), 2.44 (t, 2H, J=7.4 Hz, CH2), 2.31(q, 4H,

145

CH2) , 1.89-1.84 (m, 2H, CH2), 1.78-1.73 (m, 2H, CH2), 1.57-1.63 (m, 2H, CH2), 1.35 (s, 6H,

146

CH3) , 1.00 (t, 6H, J=6.0 Hz, CH3). 13C-NMR (100 MHz, CDCl3): ppm; 179.0 (C=O), 159.6

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(Ar-Cipso), 155.2 (Cipso), 143.2 (Ar-Cipso), 141.9 (Cipso), 131.9 (Cipso), 129.2 (Ar-CH), 127.0 (Cipso),

148

121.1 (Cipso), 115.1 (Ar-CH), 67.7 (CH2), 33.9 (CH3), 28.9 (CH2) , 25.6 (CH2), 24.5 (CH2), 17.1

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(CH2), 14.6 (CH2), 14.2 (CH3), 11.1 (CH3). HRMS (ESI): m/z: Calcd: 509.28650 [M-H]-. Found:

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509.27995.

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FTIR spectra were recorded on a Bruker Tensor 37 FT-IR spectrometer in KBr while a

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C NMR

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spectra were recorded on a Bruker Spectrospin Avance DPX400 Ultrashield spectrometer (400

155

and 100 MHz respectively) in CDCl3 and internal standard TMS. High resolution mass spectra

156

were recorded with Agilent Technologies 6224 TOF LC/MS and 6530 Accurate Mass Q-TOF

157

LC/MS, ionization method ESI.

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Varian Cary 5000 was used for UV-vis spectrophotometric measurements.

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H and

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2.3. Polyethylenimine coated magnetic nanoparticles

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Polyethylenimine (PEI) (Mw:10000 g.mol-1) was dissolved in 40 ml of Milli-Q water so that

162

the PEI concentration in the final solution is 5% (w/v). Iron(III) chloride hexahydrate

163

(FeCl3.6H2O) (2 g, 0.01 mol) and iron(II) chloride tetrahydrate (FeCl2.4H2O) (5.2 g, 0.02 mol)

164

were dissolved in this polyethyleneimine solution, respectively. The mole ratio of Fe(III)/Fe(II)

165

was kept as 2:1 while the ratio of PEI/MNP was 0.1. The solution was heated to 80 oC under

166

nitrogen atmosphere and added 50 ml of 1 M sodium hydroxide solution slow dropwise. The

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solution then was let at 80 oC for 2 h. The PEI coated magnetic Fe3O4 (PEI-Fe3O4) nanoparticles

168

separated by a centrifuge and neodymium magnet were washed many times to get rid of excess

169

polymer and base. The synthesis of the PEI-Fe3O4 nanoparticles was summarized in Scheme 1a.

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The size and morphology of the PEI-Fe3O4 nanoparticles were determined by dynamic light

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scattering (DLS, Malvern Zetasizer ZS) and transmission electron microscopy (TEM, FEI Tecnai

172

G2 F30 instrument) while the binding was characterized by FTIR and X-ray photoelectron

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spectroscopy (XPS) measurements performed by a K-Alpha-Monochromated high-performance

174

X-ray photoelectron spectrometer from VG Company. Powder X-ray diffraction (XRD)

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measurements were recorded on Micro Max 007HF DW instrument.

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2.4. Bodipy functionalized magnetic nanoparticles

Bodipy-5

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(10

mg,

0.02

mmol)

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were

dissolved

in

THF

(10

mL).

N,N-

dicyclohexylcarbodiimide (DCC) (2.9 mg, 0.01 mmol) and 4-dimethylaminopyridine (DMAP)

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(0.17 mg, 1.4 mol) were added into this solution. The PEI-Fe3O4 nanoparticles (10 mg) were

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dispersed in THF (2 mL) and added into the reaction mixture (Scheme 1b). The reaction mixture

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was bubbled under nitrogen gas for 20 minutes then let it stirring for overnight. The obtained

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Bodipy conjugated magnetic nanoparticles (BOD-MNPs) were collected via magnet and washed

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two times with water and methanol.

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2.5. Cell Culture

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Cytotoxicity of the BOD-MNPs was tested on three types of human cell line, A549 (Human

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Lung Adenocarcinoma Epithelial), Ishikawa (Human Endometrial Adenocarcinoma) and

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HUVECs (Human Umbilical Vein Endothelial Cells). A549 and HUVECs cell line were obtained

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from ATCC (American Type Cell Collection) while Ishikawa cell line was kindly provided by

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Dr. A. Bilir (Istanbul University, Turkey). A549 cells were grown in RPMI 1640 medium

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containing 10% heat-inactivated fetal calf serum (FBS), 9.2% NaHCO3 and 1%

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penicillin/streptomycin. Ishikawa cells were maintained in DMEM:F12 (1:1) medium containing

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%10 FBS, %1 penicillin-streptomycin. HUVECs were incubated and grown in Nutrient Mixture

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F12 HAM medium supplemented with 20% FBS, heparin (0.1 mg/ml), and endothelial cell

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198

growth supplement (ECGS, 0.05 mg/ml). Cells were cultured in a humidified atmosphere

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containing 5% CO2 at 37 oC. the BOD-MNPs were dispersed in DMSO with ultrasonic bath in 15

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min. and diluted in the cell culture media freshly.

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2.6. Cytotoxicity Assay

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The cytotoxicity of the BOD-MNPs was evaluated with standard MTT (3-(4,5-

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dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [22]. Cells were seeded in flat-

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bottomed 96-multiwell plates (Techno Plastic Products AG/TPP) in 100 ml media containing

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5x103 cells, and incubated at 37 C for 24 hours in a humidified atmosphere of 5% CO2 / 95% O2.

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Thereafter, old medium was replaced with fresh culture media supplemented with the BOD-

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MNPs at concentrations of 0.004, 0.008, 0.015, 0.022, 0.030, 0.037, 0.074, 0.112, 0.148 mg/ml.

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For references, old medium was replaced with fresh medium without the dye. In order to test

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cytotoxicity, the BOD-MNPs of each concentration were cultured with A549 and Ishikawa cells

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for 48h and 24h, respectively. During this period after each day, the medium was aspirated and

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100 l fresh medium containing 0.5 mg/ml MTT (Sigma) dissolved in Phosphate Buffer Saline

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(PBS) was added to culture wells. The plates with added MTT solution were then wrapped in

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aluminum foil and replaced in the 5% CO2 incubator for 2 hours. At the end of this period, the

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medium was removed and the formazan crystals formed by MTT metabolism were dissolved by

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addition of 100 l DMSO to each well. The plates were gently mixed on a plate shaker

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approximately for 5 minutes, and their absorbance values were read at 570 nm with a microtiter

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plate reader (Bio-Tek, ELX808IU, USA). Every test concentration had the eight replicates per

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assay and every experiment was carried out on at least three separate occasions. The SPSS has

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been used for the statistical analysis of assessment of the MTT assay. The data were evaluated

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using one-way ANOVA followed by the Tukey test. A value of p<0.05 was considered as

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significant.

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2.7. Fluorescence Microscopy

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Imaging and the cell uptake study were performed by fluorescence microscope (Olympus

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BX50 with U-UHK fluorescence attachment microscope). The cells were seeded on 12-well

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plates at concentration of 7x104 cells per well for 24 h and then incubated for 2 h in a media

230

containing 10% FBS and the BOD-MNPs without phenol red. Images were acquired immediately

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on live cells by fluorescence microscope. A test experiment was also design with JC-1 dye

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(common fluorescence dye) under the same conditions to compare the imaging ability of the

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BOD-MNPs according Castedo et al.s method [23].

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3. Results and discussion

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3.1. Synthesis and characterization of the Bodipy conjugated magnetic nanoparticles

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Uniform PEI coated magnetic Fe3O4 (PEI-Fe3O4) nanoparticles with narrow size

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distribution have been produced by a modified co-precipitation method in which the

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magnetic nanoparticles have been synthesized in aqueous PEI solution [24] (Scheme 1a).

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Scheme 1. Synthesis route of the PEI-Fe3O4 (a) and the BOD-MNP nanoparticles (b).

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Fig. 1A shows a TEM image of monodisperse PEI-Fe3O4 nanoparticles with a diameter of

246

about 10 nm. The size distribution of the nanoparticles was also confirmed by DLS

247

measurements. The results are also good agreement with TEM results (Fig. 1E) within

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experimental limits even though the determined mean hydrodynamic diameter of nanoparticles is

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around 21 nm. The size distribution of the BOD-MNPs was also determined whether or/not

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possible conjugation reactions result in aggregation of nanoparticles. By considering the DLS

251

results seen in Fig. S3, it was observed that the mean hydrodynamic diameter of nanoparticles

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increased to 58 nm from 21 nm as result of some aggregation of the nanoparticles by conjugation

253

of Bodipy-5. The lattice fringes of magnetic Fe3O4 nanoparticles in Fig. 1B and the electron

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diffraction (SAED) pattern in a selected area in Fig. 1D also indicates to be the crystalline form

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of Fe3O4 nanoparticles. Further compositional verification of Fe3O4 nanoparticles was proved by

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energy-dispersive X-ray (EDX) spectroscopy (Fig. 1F).

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Fig. 1. HR-TEM images and EDS spectrum of magnetic Fe3O4 nanoparticles (A,B,C: TEM

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images, E:Size distribution based on hydrodynamic radius (nm) from DLS, F:EF-

260

TEM mapping, EDS-TEM, G: EDS analysis of magnetic nanoparticles)

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261

The phase and composition of Fe3O4 nanoparticles were also determined by X-ray

262

diffraction (XRD). The diffraction pattern is consistent with the standard diffraction data of the

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cubic structure of Fe3O4 (JCPDS file No. 19-0629) [25]. These peaks, indexed to the reflections

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of (220), (311), (400), (511), (440) planes were compatible with XRD (Fig. 2) [26].

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Fig. 2. XRD pattern of magnetic Fe3O4 nanoparticles

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The existence of PEI on the Fe3O4 surfaces was confirmed by FTIR and XPS

270

measurements. The FTIR spectra on the PEI coated and bare Fe3O4 nanoparticles are seen

271

in Fig. 3A. A strong absorption peak at 583 cm-1 belongs to a characteristic band of the

272

Fe-O stretching vibrations of magnetic Fe3O4 nanoparticles [27]. This peak was shifted a

273

higher wavenumber of 591 cm-1 as a result of the PEI coating. Two peaks at 3400 and

274

1634 cm-1 are attributed to the stretching and bending vibrations of O-H bond from

275

residual water in the sample. A characteristic N-H stretching peak at 3381 cm-1 together

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with bending peaks at 894, 800 and 1551 cm-1 confirms the PEI-Fe3O4 nanoparticles [28].

277

In addition, XPS spectrum confirms the existence of PEI on the magnetic nanoparticle

278

surface, Fig. 3B. The peaks correspond to the binding energies at ~711 eV and ~725 eV

279

are related to Fe 2p3/2 and Fe 2p1/2, respectively [29]. Also, the binging energy at 531 eV is

280

attributed to O1s indicating O-Fe in magnetic phase. The peak at 402 eV corresponds to

281

N1s which proves that the magnetic Fe3O4 nanoparticles covered by PEI (Fig. 3B).

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Fig. 3. FTIR spectra (A) of the Fe3O4 nanoparticles, the PEI-Fe3O4 nanoparticles and the

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BOD-MNPs, and XPS spectrum (B) of the PEI-Fe3O4 nanoparticles

285
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In the final step, Bodipy-5 dye was attached to PEI coated magnetic nanoparticles to

288

produce the Bodipy conjugated magnetic nanoparticles (BOD-MNPs) (Scheme 1b). After

289

covalently bonding of Bodipy-5 dye to the surface of the PEI-Fe3O4 nanoparticles, EF-

290

TEM map shows boron element (green colour) on the nanoparticles (Fig. 1D). Further

291

prove of binding the Bodipy-5 ligand to the surface of PEI-Fe3O4 is given in Fig. 3A. It is

292

clear that there are some new peaks appeared corresponding to amide group after

293

covalently bonding of Bodipy-5 dye to the surface of the PEI-Fe3O4 nanoparticles. The

294

new peaks at 1658 and 1627 cm-1 are attributed to the C=O stretching and N-H bending of

295

amide bonds, respectively, which are characteristics of CONH2 group [30, 31]. In

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addition, the peak at 1446 cm-1 is due to the C-N stretching, and the 703 cm-1 band

297

represents the out of plane bending of the weak bond of N-H bond. These are typical

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Page 13 of 28

298

absorption bands of amide which prove the Bodipy dye to attach covalently to the surface

299

of the PEI-Fe3O4 nanoparticles.

300

Photophysical properties of Bodipy-5 and the BOD-MNPs were investigated by

302

measuring absorption and emission spectra. Bodipy-5 characteristically exhibits an

303

absorption peak at 497 nm (S0-S1 transition) and emission peak at 506 nm in methanol

304

solution, Fig. 4A,B. Having conjugated with the magnetic nanoparticles, the absorption

305

maximum shifts to blue region, 362 nm due to the self-absorption and scattering of light

306

caused the Fe3O4 nanoparticles (Fig. 4C). However, the fluorescent properties of the

307

BOD-MNPs are almost identical with Bodipy-5 with an emission maximum at 506 nm. It

308

is obvious that the magnetic particles do not affect the emission maximum and the BOD-

309

MNPs have a good fluorescent property even although the conjugation with the magnetic

310

particles decreases the fluorescence intensity of Bobipy-5.

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B
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312
313

Fig. 4. Absorption (A, C) and emission (B, D) spectra of Bodipy-5 and the BOD-MNPs,

314

respectively. Inset pictures: the pictures of the solution of magnetic Fe3O4 nanoparticles

315

in methanol without (1) and with (2) a neodymium magnet as well as the picture of BOD-

316

MNPs under UV-light (3).

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317

In Fig. 4, it is also seen a digital photograph of the BOD-MNPs dispersed in methanol with the

318

assistance of ultrasonic bath (Fig. 4). The nanoparticles can be easily dispersed in methanol and

319

this suspension can kept constant over a week. When a magnet placed aside, the BOD-MNPs can

320

be quickly collected in several seconds, leading to a clear solution.

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t

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Magnetic properties of uncoated Fe3O4 nanoparticles and the BOD-MNPs were performed

322

at 25 C using a VSM in an external magnetic field ranging from -100kOe to +100 kOe, as shown

324

in Fig. 5. The saturation magnetization for the Fe3O4 NPs is 76 emu/g at 298 K, and it decreased

325

after covalently bonded to Bodipy-5 dye to 60 emu/g. According to our results, such good

326

superparamagnetism will make sure that the hybrid nanoparticles can be re-dispersed rapidly as

327

soon as the magnetic field is removed, which is highly desired for their application in

328

nanomedicine.

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330
331

Fig. 5. The magnetization curves at room temperature of the PEI-Fe3O4 nanoparticles (MNPs)
(line) and the BOD-MNPs (dashed line).

332
333
334

3.2. Cytotoxicity and bio-imaging study

335
336

The cytotoxic effect of the BOD-MNPs was investigated on two different cancer cell

337

lines, A549 and Ishikawa cells as shown in Fig. S1. The cytotoxicity was also tested on

15

Page 15 of 28

healthy HUVECs for the comparison. As observed by MTT assay, all types of cells were

339

viable after 24 and 48 hours incubation with the BOD-MNPs in all tested concentrations.

340

It was observed that the BOD-MNPs did not have any cytotoxic effect to Ishikawa cells

341

even at higher concentrations whereas little toxicity of the nanoparticles on A549 cells and

342

HUVECs were observed at the studied concentrations, see Figs. S2 and S3. With this

343

respect, Ishikawa cells show different cellular response from A549 cells and HUVECs

344

under the studied concentrations. This small difference in cellular response may be

345

attributed to cell vision concept suggested by Mahmoudi et al. [32,33]. It has been

346

known that the uptake and defence mechanism could be different depending on the cell

347

type because of the cellular response against to same nanoparticles relate to the numerous

348

detoxification mechanisms [32,33]. Even though A549 cells and HUVECs have very little

349

toxicity, these findings show that the particles are quite suitable for further in vitro

350

imaging applications.

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351

The cellular internalization of the BOD-MNPs was investigated on two different

353

cancer cell lines, A549 and Ishikawa cells as shown in Fig. 6 and 7 by fluorescence

354

microscopy analysis. The uptake and cellular location was compared with depending the

355

cell type and the amount of the nanoparticles in the cell cultures. The pre-dispersed BOD-

356

MNPs were applied on the A549 and Ishikawa cells cultured in the appropriate method in

357

suitable concentrations, see Section 2.7. After 2h incubation, the images in Figs. 6 and 7

358

were taken immediately. It can be seen cellular uptake and cellular localization the BOD-

359

MNPs into the A549 cells in Fig. 6. The images taken with 40x and 100x magnification

360

are given together in Fig. 6. The BOD-MNPs were exhibited good cellular uptake and

361

there was no toxicity even at higher doses (0.148 mg/ml) at both 24 and 48 h exposures.

362

The uptake of the BODIPY-magnetic nanocomposites increases with dose dependent

363

manner. By considering all applied concentrations on A549 and Ishikawa cells, the BOD-

364

MNPs were localized in the cytoplasm within the lipophilic regions of intracellular

365

organelles with evidence for uptake in the endoplasmic reticulum. Cells were exhibited a

366

bright green cell profile. The BOD-MNPs are localized near the nucleus with increasing

367

the concentration.

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369
370

Fig. 6. Localisation and cellular uptake of the BOD-MNPs into the A549 cells. 0.037

371

mg/ml (A), 0.074 mg/ml (B), 0.112 mg/ml (C) and 0.148 mg/ml (D) of the

372

BODIPY-magnetic nanocomposite was treated in live A549 cells for 1 h. The

373

images were taken scale bar: 20 m (A-B-C-D) and 10 m (A'-B'-C'-D').

374
375

It is obvious that the imaging profile is almost the same for Ishikawa cells. Therefore

376

the images taken with the magnifications are given for only 0.037 mg/ml. for the other

377

concentration, only 40x pictures are given in Fig. 7(1). For the comparison, The A549 and

378

Ishikawa cells was dyed a standard JC-1 dye at a concentration of 2.5 g/ml. Images were

17

Page 17 of 28

379

acquired immediately on live cells by fluorescence microscope (Olympus BX50 with U-

380

UHK fluorescence attachment microscope), Fig. 7(2).

Ac
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381

382

383

Fig. 7. (1) Localisation and cellular uptake of the BOD-MNPs into the Ishikawa cells. 0.037(A)

384

and 0.074 mg/ml (B) of the dye was treated in live Ishikawa cells for 1 h. (Scale bar: 20

385

m (A-B) and 10 m (A'-B'). (2) The images of the standard JC-1 dyed A549 and

386

Ishikawa cells (Scale bar: 20 m)

387
388
389
390
391
392

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393

4. Conclusion

394

Multifunctional magnetic nanoparticles covalently conjugated with a Bodipy entity were

396

prepared by a facile method. The magnetic Fe3O4 nanoparticles coated with polyethyleneimine

397

were synthesized and then covalently conjugated a fluorophore molecule, Bodipy-5 by the

398

DCC/DMAP coupling reaction. It was found that the BOD-MNPs have no cytotoxic effect to

399

A549 and Ishikawa cells as well as HUVECs. In vitro bio-imaging results revealed that the BOD-

400

MNPs can easily penetrate into the cell cytoplasm in the living cells with their own fluorescence

401

properties. For this reason, these are potentially capable of cell imaging. In addition, their ability

402

to accumulate in cancer cells besides their superparamagnetic properties makes them a potential

403

MRI contrast agent.

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405

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Acknowledgements

406

The authors acknowledge the Akdeniz University Coordination Unit of Scientific Research

408

Projects (project No. 2012.01.0115.001) for their financial support. The authors are grateful to

409

Prof. Engin U. Akkaya (Department of Chemistry, Bilkent University, Ankara-Turkey) for

410

laboratory facilities. The authors thank especially Mustafa Gler (UNAM, Bilkent University,

411

Ankara-Turkey) for technical assistance with the HR-TEM measurements.

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413

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473

Figure Captions

474

Fig. 1. HR-TEM images and EDS spectrum of magnetic Fe3O4 nanoparticles (A,B,C: TEM

476

images, E:Size distribution based on hydrodynamic radius (nm) from DLS, F:EF-TEM

477

mapping, EDS-TEM, G: EDS analysis of magnetic nanoparticles)

ip
t

475

Fig. 2. XRD pattern of magnetic Fe3O4 nanoparticles

479

Fig. 3. FTIR spectra (A) of the Fe3O4 nanoparticles, the PEI-Fe3O4 nanoparticles and the BOD-

cr

478

MNPs, and XPS spectrum (B) of the PEI-Fe3O4 nanoparticles

480

Fig. 4. Absorption (A, C) and emission (B, D) spectra of Bodipy-5 and the BOD-MNPs,

482

respectively. Inset pictures: the pictures of the solution of magnetic Fe3O4 nanoparticles

483

in methanol without (1) and with (2) a neodymium magnet as well as the picture of

484

BOD-MNPs under UV-light (3).

an

Fig. 5. The magnetization curves at room temperature of the PEI-Fe3O4 nanoparticles (MNPs)

485

us

481

(line) and the BOD-MNPs (dashed line).

486

Fig. 6. Localisation and cellular uptake of the BOD-MNPs into the A549 cells. 0.037 mg/ml

488

(A), 0.074 mg/ml (B), 0.112 mg/ml (C) and 0.148 mg/ml (D) of the BODIPY-

489

magnetic nanocomposite was treated in live A549 cells for 1 h. The images were

Ac
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487

taken scale bar: 20 m (A-B-C-D) and 10 m (A'-B'-C'-D').

490
491

Fig. 7. (1) Localisation and cellular uptake of the BOD-MNPs into the Ishikawa cells. 0.037(A)

492

and 0.074 mg/ml (B) of the dye was treated in live Ishikawa cells for 1 h. (Scale bar: 20

493

m (A-B) and 10 m (A'-B'). (2) The images of the standard JC-1 dyed A549 and

494

Ishikawa cells (Scale bar: 20 m)

495
496
497
498
499

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ip
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500
501
502
503
504
505
506

Fig. 1

507
508
509
510

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ip
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511

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512
513
514
515

Fig. 2

516

517

Ac
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518
519
520
521
522
523
524
525
526
527
528

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Page 24 of 28

529

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(A)

530

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(B)

531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549

Fig. 3

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Page 25 of 28

550

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551
552

Fig. 4

553

Ac
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554

555
556
557

Fig. 5

558

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Page 26 of 28

ip
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Ac
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559
560
561
562
563
564
565
566

Fig. 6

567
568
569
570
571
572

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Ac
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574
575

576
577
578
579
580
581
582
583

Fig. 7

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