Synthesis characterization of near-infrared fluorescent andmagnetic iron zero-valent nanoparticles

Synthesis and characterization of near-infrared ?uorescent and magnetic iron zero-valent nanoparticles

Nagore Pérez a ,Leire Ruiz-Rubio a ,*,JoséLuis Vilas a ,b ,Matilde Rodríguez a ,Virginia Martinez-Martinez a ,Luis M.León a ,b

a

Departamento de Química Física,Facultad de Ciencia y Tecnología,Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU),Apdo 644,Bilbao 48080,Spain b

Basque Center for Materials,Applications and Nanoestructures (BCMATERIALS)Parque Tecnológico de Bizkaia,Ed 500,Derio 48160,Spain

A R T I C L E I N F O

Article history:

Received 4May 2015

Received in revised form 4September 2015Accepted 6September 2015

Available online 9September 2015

Keywords:

Iron zero valent nanoparticles Fluorescent Magnetic

Polyethylenglycol

A B S T R A C T

Polyethylene glycol coated iron nanoparticles were synthesized by a microemulsion method,modi ?ed and functionalized.The polymer coating has a crucial role,preventing the iron oxidation and allowing the functionalization of the particles.The nanoparticles were characterized and their magnetic properties studied.A photochemical study of the iron nanoparticles conjugated with a near-infrared ?uorescent dye,Alexa Fluor 660,con ?rmed that the ?uorescent dye is attached to the nanoparticles and retains its ?uorescent properties.The bioimages in red and near-infrared (NIR)region are favourable due to its minimum photodamage and deep tissue penetration.The nanoparticles obtained in this study present a good magnetic and ?uorescent properties being of particular importance for potential applications in bioscience.

?2015Elsevier B.V.All rights reserved.

1.Introduction

A broad range of nanosized inorganic particles,including magnetic nanoparticles and quantum dots,have been extensively investigated because of their unique optical,electrical and magnetic properties [1–5].Moreover,magnetic iron oxide colloids have been successfully used as magnetic resonance imaging (MRI)contrast agents and for cancer hyperthermia therapy [6–9].

The shape,size and size distribution of the magnetic materials are the key factors in determining their chemical and physical properties.Thus,the development of size and shape-controlled magnetic materials is crucial for their application [3,9].So far,the most widely used and studied magnetic material is iron oxide,in the form of magnetite (Fe 3O 4)and maghemite (g -Fe 2O 3).Elemental iron has a signi ?cantly higher magnetic moment than its oxides.Moreover,elemental iron is the most useful among the ferromagnetic elements;it has the highest magnetic moment at room temperature (218emu g à1in bulk),and a Curie temperature which is high enough for the majority of practical applications.However,obtaining Fe nanoparticles,relatively free of oxide (usually Fe 3O 4),is still a challenge,to a large extent,not overcome [10–13].

Besides the properties of the metallic core,the coating of the nanoparticles could determinate or improve the uses of this kind of materials.For example,functionalized magnetic nanoparticles have been employed for site-speci ?c drug delivery [14]or treatment waterwaste [15,16].The variety of potential coating materials is continuously increasing with the development of new polymeric materials.However,polyethylene glycol (PEG)could be considered one of the most suitable polymer coatings for nanoparticles designed to be used in biomedicine.PEG is a water-soluble polymer with a low toxicity and antibiofouling properties that make it an appropriate candidate for several bioscience related applications [17,18].PEG chains attached to a nanoparticle surface exhibit a rapid chain motion,this could contribute to the good physiological properties of the PEGylated nanoparticles [19]for imagining and therapy application.Also,successful studies haven been devoted to PEG-PLA coated nano-particles for drug delivery [20,21].PEG grafted onto the surface of nanoparticles provides steric stabilization that competes with the destabilizing effects of Van der Waals and magnetic attraction energies.Thus,there is a growing demand for improved methods for the synthesis and characterization of polyethylene glycol (PEG)derivatives [22–25].Especially,polyethylene glycols (PEGs)of long polymeric chains have found signi ?cant applications in the structure stabilization [26–28].

Finally,the polymeric coatings of the nanoparticles could be conjugated with antibodies or ?uorescent dyes adding different

*Corresponding author.

E-mail address:leire.ruiz@ehu.eus (L.Ruiz-Rubio).

https://www.360docs.net/doc/fa5459080.html,/10.1016/j.jphotochem.2015.09.0041010-6030/?2015Elsevier B.V.All rights reserved.

Journal of Photochemistry and Photobiology A:Chemistry 315(2016)1–7

Contents lists available at ScienceDirect

Journal of Photochemistry and Photobiology A:

Chemistry

j o u rn a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j p h o t o c h e

m

properties to the system[29–31].That is,?uorescent-magnetic nanoparticles could be designed as an all-in-one diagnostic and therapeutic tool,able to visualize and simultaneously treat various diseases.

Fluorescence imaging is one of the most powerful techniques for monitoring biomolecules in living https://www.360docs.net/doc/fa5459080.html,pared with ?uorescent imaging in the visible region,biological imaging in red and near-infrared(NIR)region is favourable due to its minimum photodamage,deep tissue penetration,and minimum background auto?uorescence caused by biomolecules in living systems. Therefore,chromophores with emission in red or near-infrared region have been paid increasing attention in recent years[32,33].

However,there is a speci?c dif?culty in the preparation of ?uorescent magnetic nanoparticles due to the risk of quenching of the?uorophore on the particle surface by the magnetic core.This problem could be solved by coating the magnetic core with a stable isolating shell prior to the introduction of the?uorescent molecule or by attaching an appropriate spacer to the?uorophore.Most ?uorescent magnetic nanoparticles thus have a core-shell struc-ture.

Several studies have been devoted to develop iron oxide nanoparticles conjugated with?uorescent dyes,in order to obtain dual-responsive nanoparticles,with magnetic and?uorescent response[31].Often,the methods are time consuming due to the many synthetic steps or the fact that gold or silica precoating is required to protect the iron oxide nanoparticles previous to their functionalization[34–36].Also,there is a signi?cant lack on studies about iron nanoparticles functionalized with?uorophores [37].The aim of this work is to synthesize iron nanoparticles coated with a PEG-derivative and functionalized with a?uorescent dye.The iron core of the nanoparticles will provide higher magnetization saturation than iron oxides,the PEG not only protects the metallic core but also adds interesting properties to biologically related applications.The selected?uorescent dye, imaging in red and near-infrared,is highly adequate for an application in medicine owing to its low photodamage.So,the obtained nanoparticles could be highly promising materials for combined MR/Optical imaging applications.

2.Materials and methods

2.1.Chemicals

All chemicals were reagent grade and used without puri?cation. Ferrous chloride tetrahydrate(FeCl2á4H2O),sodium borohydride (NaBH4)and cyclohexan solvent were purchased from Sigma–Aldrich.Methanol and chloroform were purchased from Panreac and Lab-Scan,respectively.Polyethylene glycol(PEG)of1000g molà1molecular weight and methoxy polyethylene glycol(mPEG) of2000g molà1molecular weight were obtained from Sigma–Aldrich.Deionized Millipore Milli-Q water was used in all experiments.Alexa Fluor1660Protein Labeling Kit was purchased from Invitrogen.

2.2.Synthesis of iron nanoparticles

The preparation of PEG-stabilized nanoscale zero-valent iron nanoparticles was carried out via a controlled microemulsion method.The microemulsion synthetic methodology makes use of a biphasic heterogeneous solution of water-in-oil in which iron precursors are stirred.Water droplets are used as nucleation sites for the formation of nanoparticles,often in the presence of surfactant molecules dispersed in the oil,essentially forming micelles.

The reactions were carried out at room temperature using a single micellar system(sample FePEG-04)and two micellar systems(sample FePEG-02).The procedure followed in the?rst

case is described here.

A surfactant solution prepared by dissolving31.5g of polyeth-

ylene glycol in105mL of cyclohexane was maintained under

stirring and degassed for10min under N2atmosphere.Next,6mL

of0.33M FeCl2á4H2O were added to the surfactant solution,stirred and degassed for10min.Metal particles were formed inside the

reverse micelles via reduction of the metal salt using an excess of

NaBH4(6mL, 1.76M).After a few minutes,the reaction was

quenched by adding50mL of chloroform and50mL of methanol.

The black precipitate was recovered with a permanent magnet,

washed several times with methanol and dried under vacuum.

The same procedure was carried out in the synthesis performed

by two micellar systems with the only difference that the reducing

agent(NaBH4),was added in aqueous solution instead of in solid

form.This solution,when added to?ask reaction,will result the

second micellar system.De?nitely,the method involves mixing

two microemulsions:one containing the metal salt and the other

the reducing agent;due to collision and coalescence of the droplets

the reactants are brought into contact and react to form the

nanoparticles.

Polyethylene glycol methyl ether(mPEG)shows greater

versatility in functionalization,which increases the potential

applications of nanoparticles.Speci?cally,this will be the

derivative chosen to functionalize nanoparticles.The syntheses

with this surfactant were carried out at room temperature using a

single-micellar system,0.40g of iron salt,0.20g of the reducing

agent,105cm3of cyclohexane and6.0g of water.The concentration

of surfactant in this system was0.095M.

2.3.Functionalization of nanoparticles and labelling with?uorescent

dye

The incorporation of the?uorescent molecule to the nano-

particles consists of several steps.Firstly,functionalized nano-

particles are synthesized and then the?uorophore is anchored.

After that the labelled nanoparticles must be puri?ed to take out

the excess dye by size-exclusion chromatography.

2.3.1.Modi?cation of mPEG

Polyethylene glycol methyl ether(mPEG)of molecular weight

2000g molà1was?rstly treated to obtain the aldehyde-derivative

by oxidation of the hydroxyl end groups by dimethylsulfoxide

(DMSO)and acetic anhydride at room temperature.Then the

m-PEG-amine was obtained by the method described by Harris

et al.[38],via reduction of the aldehyde groups using sodium

cyanoborohydride in methanol at room temperature.

2.3.2.Synthesis of nanoparticles with mPEG-NH2and PEG

The synthesis of nanoparticles was performed by the method

previously described for one micellar system.Owing to the small

amount of material?uorescent necessary,the appropriate amount

of mPEG-NH2was used,and the rest was PEG surfactant,as already

shown,to provide adequate protection to the nanoparticles.

The surfactant consisted of a mixture of7.5g of PEG and217mg

of mPEG-NH2,amounts required to have a total surfactant

concentration of0.30M.

https://www.360docs.net/doc/fa5459080.html,belling of nanoparticles

The interaction of metal nanoparticles with?uorophores near

its surface affects the intensity of their emission being critical the

distance between the?uorophore and the surface of the

nanoparticle so that the?uorescence is quenched when the

distance is too short.For this study Alexa Fluor660was used.This

is a succinimidyl ester of Alexa Fluor which exhibits bright ?uorescence and high photostability characteristics allowing us to

2N.Pérez et al./Journal of Photochemistry and Photobiology A:Chemistry315(2016)1–7

capture images that were previously unattainable with conven-tional ?uorophores.Moreover it provides an ef ?cient and convenient way to selectively link to primary amines.On the other hand,its absorption and ?uorescence bands are far from those of the nanoparticles,so that the spectral overlapping is negligible.

The PEGylated nanoparticles were ?uorescently labelled by reaction with Alexa Fluor 660carboxylic acid succinimidyl ester which formed a chemical bond with the NH 2group of mPEG-NH 2.For that,the procedure established by Invitrogen [39]was followed.Brie ?y,a solution of sodium bicarbonate was added to the nanoparticles suspension in order to reach a pH between 7,5and 8,5since succinimidyl esters react ef ?ciently at this pH range.The reactive dye was added to the solution and the reaction mixture was stirred for 1h at room temperature.

Separation of the labelled nanoparticles from dye which has remained unreacted was carried out using a puri ?cation column containing the Bio-Rad BioGel P30resin.

2.4.Characterization of nanoparticles

The crystallite phase of the coated nanoparticles was identi ?ed by recording X-ray diffraction patterns (XRD)using a Bragg –Brentano u /2u Philips diffractometer.

Size and shape of nanoparticles were studied by transmission electron microscopy (TEM).Measurements were carried out using a Philips CM 200equipment operating at an accelerating voltage of 200KV.For this,a drop of dilute methanol solution of the nanoparticles was placed onto a copper grid coated with carbon ?lm with a Formvar membrane and allowed to air dry before being inserted into the microscope.

Magnetic properties were studied with a vibrating sample magnetometer (VSM).

57

Fe M?ssbauer spectroscopy measurements were carried out at room temperature (RT)in transmission geometry using a conventional spectrometer with a 57Co-Rh source.Reported isomer shift (d )and internal magnetic hyper ?ne ?eld (BHF)values are relative to metallic Fe at room temperature.

The UV –vis absorption spectra were recorded on a Varian double beam spectrophotometer (Cary 4E)in transmittance mode,in the region of 200–900nm.

The ?uorescence spectra were performed on a SPEX ?uorimeter (Fluorolog 3-22).The emission spectra were recorded in the 250–800nm range,by exciting at different wavelengths,depend-ing on the sample.

Fluorescence single-particle measurements were performed in a time-resolved ?uorescence confocal microscope (model Micro

Time 200,PicoQuant).Fluorescence lifetime images (FLIM)are processed with ShymPhotime software (Picoquant)by sorting all photons of one pixel into a histogram and ?tted to an exponential decay function to extract lifetime information;the procedure was repeated for every pixel in the image.A 640nm pulsed laser diode,with 70ps pulses was used as excitation source.Spectra were recorded by directing the emission beam to an exit port,where a spectrograph (model Shamrock 300mm)coupled to a CCD camera (Newton EMCCD 1600?200,Andor)were mounted.

3.Results and discussion

3.1.Spectroscopic and crystallographic characterization

Polyethylene glycol and polyethylene glycol methyl ether coated iron nanoparticles were characterized by XRD measure-ments as shown in Fig.1.The spectrum of PEG coated samples obtained by one or two micellar systems (Fig.1a)shows three characteristic broad peaks at 2u =44.81 ,65.07 and 82.49 ,which correspond to the (110),(200),and (211)families of planes of the bcc lattice reported for the a -Fe phase.The dimension of the crystallites,D hkl ,was estimated by Scherrer equation in 27.8nm.The nanoparticles obtained with mPEG as surfactant present a diffractogram with a peak of high intensity at 2u =45 ,corre-sponding to the bcc lattice (Fig.1b).This kind of diffractogram is characteristic of samples with low crystallinity and very polydis-perse sizes.

From TEM images and histograms (Fig.2),it can be concluded that each Fe/PEG unit consists in a spherical Fe core with an average size of 3.8nm and its own polymeric coating of about 6nm.According to XRD results,the FemPEG-01sample was very polydisperse and it was very dif ?cult to obtain a mean diameter.In general,the size of the nanoparticles was between 10and 20nm.

The values obtained are similar to those obtained when using nonylphenypentaethoxylated (NP5)[40]as surfactant whose value was around 10nm (Fe core 7.5nm and polymeric shell 2.8nm).PEG provides a thicker coating shell than NP5,probably due to the different molecular weight of both surfactants.

3.2.Magnetic properties

Magnetization vs applied ?eld hysteresis loops were measured using VSM to assess the magnetic properties of the synthesized nanoparticles.The saturation magnetization values were normal-ized to the mass of nanoparticles to yield the speci ?c magnetiza-tion,M s (emu g à1

).

Fig.1.X-ray diffractograms of the synthesized iron nanoparticles:(a)Polyethylenglycol coated samples and (b)polyethylene glycol methyl ether coated sample.

N.Pérez et al./Journal of Photochemistry and Photobiology A:Chemistry 315(2016)1–7

3

Fig.3shows the magnetic hysteresis loops of the samples at room temperature.The saturation magnetization of FePEG nano-particles is shown in Table 1.The saturation magnetization arises from both the iron core (218emu g à1),and the iron oxide shell (for Fe 3O 480–92emu g à1),based on the relative weight percentage of iron,iron oxide and non-magnetic coatings on the particle surface.For particles having a similar shell thickness,the weight ratio of the iron core to the iron oxide shell is greater for large particles than for small particles.All the samples have coercitivity less than 15mT and a remanence less than 25A m 2kg à1.This suggested that the particles could aggregate after the removal of the external ?eld due to the remaining magnetization.

57

Fe M?ssbauer spectroscopy measurements were carried out for the FePEG-04sample due to it has the best magnetic

properties

Fig.2.Micrographs of (a)FePEG-02,(b)FePEG-04and (c)FemPEG-01

samples.

Fig.3.Magnetization curves.

Table 1

Saturation magnetization (Ms),coercitive ?eld (Hc)and remanent magnetization.

Muestra

Ms (A m 2kg à1)

Hc (mT)

Mr (A m 2kg à1)

FePEG-0211615.319.9FePEG-0413513.221.3FemPEG-01

108

16.8

19.2

Fig.4.RT M?ssbauer spectrum for FePEG-04sample.

4

N.Pérez et al./Journal of Photochemistry and Photobiology A:Chemistry 315(2016)1–7

of the studied samples(Fig.4).The RT M?ssbauer spectrum qualitatively consist in a sextet(62%of the total area),attributed to bcc Fe(BHF=32.89T and d=à0.106mm sà1)coupled to a doublet corresponding to Fe2+or Fe3+.The appearance of both signals would indicate the occurrence of an oxidation process leading to the formation of magnetite(Fe3O4).Any other ordered phase is not observed since more sextets were not found.The iron oxides present in these samples are not magnetically ordered due to the absence of further sextets.This was con?rmed by the XPS(Apendix A,Fig.S5)where the peaks at710.30,718.98(small peak)and 723.32eV represent the binding energies of Fe(2p3/2)shake-up satellite2p3/2and2p1/2,respectively.In addition,a small shoulder at705,87eV suggest the peak of2p3/2of zero-valent iron[41].

All the studied systems present a high reproducibility as could be con?rm in the supporting information(Supporting information (Appendix A))in which the obtained X-ray difratograms and magnetization curves are shown.

3.3.Fluorescent measurements

In this section the photophysical study of the nanoparticles

conjugated with the?uorescent dye is described.Fig.5shows the height-normalized absorption spectrum of the Alexa Fluor1 660and the labelled sample.As can be seen,the absorption spectra are almost identical and show the principal absorption band centred at668nm,indicating the presence of the dye in the nanoparticles.Furthermore,a weak band in the UV region of the spectrum,around250nm,could include iron oxides such as hematite,magnetite or maghemite[42].

Fig.6shows the height-normalized?uorescence spectra of the fraction with the highest content of nanoparticles with dye in suspension at two excitation wavelengths,250and620nm.On the one hand,when the excitation of the sample takes place directly to the absorption band of the dye(620nm,see Fig.5)the emission band is obtained at696nm,emission band typical of Alexa Fluor 6601dye,indicating its presence in the particles.In order to compare the?uorescence ef?ciency of Alexa660dye in solution and anchored at the nanoparticles,the ratio between the ?uorescence intensity and the absorbance of the sample at the excitation wavelength is analysed(Fig.S6).In this way and assuming a quantum yield of around0.37for Alexa660in aqueous solution[36],an estimated quantum yield of around0.13is obtained for the dye at the nanoparticles in suspension On the other hand,when the excitation wavelength was?xed at250nm (absorption attributed mainly to the iron oxides present in the nanoparticles)the obtained band at390nm can be attributed to the typical emission of nanoparticles of iron oxide present in the sample.In addition,the dye emission band is also present.

Although the absorption and?uorescence spectroscopic

techniques indicate the presence of?uorescence dye in the

suspension of nanoparticles,to con?rm the anchorage to the

nanoparticles surface confocal?uorescence time resolved micros-

copy measurements were carried out.This technique allows the

study of the?uorescent properties of the dye anchored onto single

nanoparticles[43].In this way it can be obtained information

about lifetimes of a single particle(Fig.7),and also,through a CCD

camera,a spectrum of the?uorescence in single particle can be

obtained(Fig.8).

So,by positioning the excitation laser(640nm)in the centre of

each nanoparticle,the?uorescence spectrum of the anchored dye

nanoparticle is obtained(Fig.8).In addition,the?gure includes the

spectrum of dye in solution measured at the same conditions.The

maximum of?uorescence are696nm for dye and687nm for the

dye anchored to nanoparticles.The displacement of the maximum

towards lower wavelength,is a typical effect of dyes adsorbed in

surfaces,as the case of the iron nanoparticles.

Fig.9shows the?uorescence decay curves obtained by confocal

microscopy for the dye in solution and labelled dye in each

nanoparticle and respective histograms.

The half lifetime of free dye presents monoexponencial

behaviour,with a value of?uorescence life time t=1.8ns,while the conjugated nanoparticles presents a biexponencial behaviour

with:life time t1%0.1–0.5ns y t2=1.5–1.7ns(Fig.9).These values have been obtained after the analysis of,at least,10individual

particles.

The short half lifetime,around0.1–0.6ns can be attributed to

the light scattered by the nanoparticle itself and the obtained long

half life time(t2=1.5–1.7)is attributed to anchored dye to nanoparticle surface.

A

b

s

o

r

b

a

n

c

e

Wavelength (nm)

Fig.5.Height-notmalized absorption spectra of Alexa Fluor660dye and iron

labeled nanoparticles in aqueous buffer suspension.

F

l

u

o

r

e

s

c

e

n

c

e

I

n

t

e

n

s

i

t

y

(

a

.

u

.

)

Wavelength (nm)

Fig.6.Height-normalized?uorescence spectra of iron nanoparticles in aqueous

buffer suspension at excitation wavelengths of250and620nm.

Fig.7.Fluorescence microscopy image of single particles.

N.Pérez et al./Journal of Photochemistry and Photobiology A:Chemistry315(2016)1–75

The slight decrease of the long lifetime of anchored dye regarding the diluted suspension of the nanoparticle can be attributed to the dye quenching due to the presence of iron oxide.

Confocal ?uorescence microscopy con ?rmed that the dye is labelled onto nanoparticles and maintains its ?uorescent proper-ties.Therefore,the trajectory of these nanoparticles may be monitored by ?uorescence microscopy under red excitation in vitro or in vivo experiments.

4.Conclusions

In this study,iron nanoparticles coated with PEG and mPEG were prepared and characterized.The nanoparticles present high magnetic susceptibility and sizes between 10and 15nm.It is noteworthy that the synthesized nanoparticles are mainly zero-valent iron.

The FemPEG nanoparticles were successfully functionalized and conjugated with a ?uorescent dye.Thus,amine-reactive N -hydroxysuccinimidyl ester of Alexa Fluor 660dye was conju-gated to the nanoparticle surface.This dye produces bright far red ?uorescence emission with a peak at 690nm under red excitation light (in the clinic window).

Studies of confocal ?uorescence microscopy con ?rmed that the ?uorescent dye is attached to the nanoparticles and retains its

?uorescent properties which could make possible to monitor the course of in vitro or in vivo samples using ?uorescent microscopy red under excitation.

The magnetic properties of synthesized nanoparticles added to its ?uorescent response result in a suitable material for be detected by both magnetic and ?uorescent techniques for combined MR/Optical imaging applications.

Acknowledgements

Authors thank the Basque Country Government for ?nancial support (ACTIMAT project,ETORTEK programme IE10-272)(Ayu-das para apoyar las actividades de los grupos de investigación del sistema universitario vasco,IT718-13and IT339-10).Technical and human support provided by SGIKER (UPV/EHU,MICINN,GV/EJ,ERDF and ESF)is gratefully acknowledged.V.M.M.acknowledges the Ramon y Cajal contract with the Ministerio de Economía y Competitividad,(RYC-2011-09505).

Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at https://www.360docs.net/doc/fa5459080.html,/10.1016/j.jphotochem.2015.09.004.

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Fig.8.Fluorescence spectrum of a single particle (red curve)and a diluted dye solution (black curve)registered in time-resolved ?uorescence confocal microscope at excitation wavelength of 640nm.(For interpretation of the references to colour in this ?gure legend,the reader is referred to the web version of this

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