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Synthesis and characterization of near-infrared fluorescent and magnetic iron zero-valent nanoparticles
Journal of Photochemistry and Photobiology A: Chemistry 315 (2016) 1–7
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Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
Synthesis and characterization of near-infrared fluorescent and magnetic iron zero-valent nanoparticles Nagore Péreza , Leire Ruiz-Rubioa,* , José Luis Vilasa,b , Matilde Rodrígueza , Virginia Martinez-Martineza , Luis M. Leóna,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
A B S T R A C T
Article history: Received 4 May 2015 Received in revised form 4 September 2015 Accepted 6 September 2015 Available online 9 September 2015
Polyethylene glycol coated iron nanoparticles were synthesized by a microemulsion method, modified 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 fluorescent dye, Alexa Fluor 660, confirmed that the fluorescent dye is attached to the nanoparticles and retains its fluorescent 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 fluorescent properties being of particular importance for potential applications in bioscience. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Iron zero valent nanoparticles Fluorescent Magnetic Polyethylenglycol
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 (Fe3O4) and maghemite (g-Fe2O3). Elemental iron has a significantly 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 (218 emu g 1 in bulk), and a Curie temperature which is high enough for the majority of practical applications. However, obtaining Fe nanoparticles, relatively free of oxide (usually Fe3O4), is still a challenge, to a large extent, not overcome [10–13].
* Corresponding author. E-mail address: [email protected] (L. Ruiz-Rubio). http://dx.doi.org/10.1016/j.jphotochem.2015.09.004 1010-6030/ ã 2015 Elsevier B.V. All rights reserved.
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-specific 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 nanoparticles 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 significant applications in the structure stabilization [26–28]. Finally, the polymeric coatings of the nanoparticles could be conjugated with antibodies or fluorescent dyes adding different
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properties to the system [29–31]. That is, fluorescent-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 systems. Compared with fluorescent 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 autofluorescence 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 specific difficulty in the preparation of fluorescent magnetic nanoparticles due to the risk of quenching of the fluorophore 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 fluorescent molecule or by attaching an appropriate spacer to the fluorophore. Most fluorescent magnetic nanoparticles thus have a core-shell structure. Several studies have been devoted to develop iron oxide nanoparticles conjugated with fluorescent dyes, in order to obtain dual-responsive nanoparticles, with magnetic and fluorescent 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 significant lack on studies about iron nanoparticles functionalized with fluorophores [37]. The aim of this work is to synthesize iron nanoparticles coated with a PEG-derivative and functionalized with a fluorescent 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 fluorescent 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.
systems (sample FePEG-02). The procedure followed in the first case is described here. A surfactant solution prepared by dissolving 31.5 g of polyethylene glycol in 105 mL of cyclohexane was maintained under stirring and degassed for 10 min under N2 atmosphere. Next, 6 mL of 0.33 M FeCl24H2O were added to the surfactant solution, stirred and degassed for 10 min. Metal particles were formed inside the reverse micelles via reduction of the metal salt using an excess of NaBH4 (6 mL, 1.76 M). After a few minutes, the reaction was quenched by adding 50 mL of chloroform and 50 mL 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 flask reaction, will result the second micellar system. Definitely, 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. Specifically, 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, 0.40 g of iron salt, 0.20 g of the reducing agent, 105 cm3 of cyclohexane and 6.0 g of water. The concentration of surfactant in this system was 0.095 M.
2. Materials and methods
2.3.1. Modification of mPEG Polyethylene glycol methyl ether (mPEG) of molecular weight 2000 g mol 1 was firstly 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.
2.1. Chemicals All chemicals were reagent grade and used without purification. Ferrous chloride tetrahydrate (FeCl24H2O), 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) of 1000 g mol 1 molecular weight and methoxy polyethylene glycol (mPEG) of 2000 g mol 1 molecular weight were obtained from Sigma– Aldrich. Deionized Millipore Milli-Q water was used in all experiments. Alexa Fluor1660 Protein 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
2.3. Functionalization of nanoparticles and labelling with fluorescent dye The incorporation of the fluorescent molecule to the nanoparticles consists of several steps. Firstly, functionalized nanoparticles are synthesized and then the fluorophore is anchored. After that the labelled nanoparticles must be purified to take out the excess dye by size-exclusion chromatography.
2.3.2. Synthesis of nanoparticles with mPEG-NH2 and PEG The synthesis of nanoparticles was performed by the method previously described for one micellar system. Owing to the small amount of material fluorescent necessary, the appropriate amount of mPEG-NH2 was used, and the rest was PEG surfactant, as already shown, to provide adequate protection to the nanoparticles. The surfactant consisted of a mixture of 7.5 g of PEG and 217 mg of mPEG-NH2, amounts required to have a total surfactant concentration of 0.30 M. 2.3.3. Labelling of nanoparticles The interaction of metal nanoparticles with fluorophores near its surface affects the intensity of their emission being critical the distance between the fluorophore and the surface of the nanoparticle so that the fluorescence is quenched when the distance is too short. For this study Alexa Fluor 660 was used. This is a succinimidyl ester of Alexa Fluor which exhibits bright fluorescence and high photostability characteristics allowing us to
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capture images that were previously unattainable with conventional fluorophores. Moreover it provides an efficient and convenient way to selectively link to primary amines. On the other hand, its absorption and fluorescence bands are far from those of the nanoparticles, so that the spectral overlapping is negligible. The PEGylated nanoparticles were fluorescently labelled by reaction with Alexa Fluor 660 carboxylic acid succinimidyl ester which formed a chemical bond with the NH2 group of mPEG-NH2. For that, the procedure established by Invitrogen [39] was followed. Briefly, a solution of sodium bicarbonate was added to the nanoparticles suspension in order to reach a pH between 7,5 and 8,5 since succinimidyl esters react efficiently at this pH range. The reactive dye was added to the solution and the reaction mixture was stirred for 1 h at room temperature. Separation of the labelled nanoparticles from dye which has remained unreacted was carried out using a purification column containing the Bio-Rad BioGel P30 resin. 2.4. Characterization of nanoparticles The crystallite phase of the coated nanoparticles was identified 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 200 equipment operating at an accelerating voltage of 200 KV. For this, a drop of dilute methanol solution of the nanoparticles was placed onto a copper grid coated with carbon film 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 Mössbauer 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 hyperfine field (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 200–900 nm. The fluorescence spectra were performed on a SPEX fluorimeter (Fluorolog 3-22). The emission spectra were recorded in the 250–800 nm range, by exciting at different wavelengths, depending on the sample. Fluorescence single-particle measurements were performed in a time-resolved fluorescence confocal microscope (model Micro
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Time 200, PicoQuant). Fluorescence lifetime images (FLIM) are processed with ShymPhotime software (Picoquant) by sorting all photons of one pixel into a histogram and fitted to an exponential decay function to extract lifetime information; the procedure was repeated for every pixel in the image. A 640 nm pulsed laser diode, with 70 ps pulses was used as excitation source. Spectra were recorded by directing the emission beam to an exit port, where a spectrograph (model Shamrock 300 mm) 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 measurements 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 (11 0), (2 0 0), and (2 11) families of planes of the bcc lattice reported for the a-Fe phase. The dimension of the crystallites, Dhkl, was estimated by Scherrer equation in 27.8 nm. The nanoparticles obtained with mPEG as surfactant present a diffractogram with a peak of high intensity at 2u = 45 , corresponding to the bcc lattice (Fig. 1b). This kind of diffractogram is characteristic of samples with low crystallinity and very polydisperse 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 3.8 nm and its own polymeric coating of about 6 nm. According to XRD results, the FemPEG-01 sample was very polydisperse and it was very difficult to obtain a mean diameter. In general, the size of the nanoparticles was between 10 and 20 nm. The values obtained are similar to those obtained when using nonylphenypentaethoxylated (NP5) [40] as surfactant whose value was around 10 nm (Fe core 7.5 nm and polymeric shell 2.8 nm). 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 field hysteresis loops were measured using VSM to assess the magnetic properties of the synthesized nanoparticles. The saturation magnetization values were normalized to the mass of nanoparticles to yield the specific magnetization, Ms (emu g 1).
Fig. 1. X-ray diffractograms of the synthesized iron nanoparticles: (a) Polyethylenglycol coated samples and (b) polyethylene glycol methyl ether coated sample.
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Fig. 2. Micrographs of (a) FePEG-02, (b) FePEG-04 and (c) FemPEG-01 samples.
Fig. 3 shows the magnetic hysteresis loops of the samples at room temperature. The saturation magnetization of FePEG nanoparticles is shown in Table 1. The saturation magnetization arises from both the iron core (218 emu g 1), and the iron oxide shell (for Fe3O4 80–92 emu g 1), 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 15 mT and a remanence less than 25 A m2 kg 1. This suggested that the particles could aggregate after the removal of the external field due to the remaining magnetization. 57 Fe Mössbauer spectroscopy measurements were carried out for the FePEG-04 sample due to it has the best magnetic properties
Fig. 3. Magnetization curves.
Table 1 Saturation magnetization (Ms), coercitive field (Hc) and remanent magnetization. Muestra
Ms (A m2 kg
FePEG-02 FePEG-04 FemPEG-01
116 135 108
1
)
Hc (mT)
Mr (A m2 kg
15.3 13.2 16.8
19.9 21.3 19.2
1
)
Fig. 4. RT Mössbauer spectrum for FePEG-04 sample.
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exc 250 exc 620
Fluorescence Intensity (a.u.)
of the studied samples (Fig. 4). The RT Mössbauer spectrum qualitatively consist in a sextet (62% of the total area), attributed to bcc Fe (BHF = 32.89 T and d = 0.106 mm s 1) 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 confirmed by the XPS (Apendix A, Fig. S5) where the peaks at 710.30, 718.98 (small peak) and 723.32 eV represent the binding energies of Fe (2p3/2) shake-up satellite 2p3/2 and 2p1/2, respectively. In addition, a small shoulder at 705,87 eV suggest the peak of 2p3/2 of zero-valent iron [41]. All the studied systems present a high reproducibility as could be confirm in the supporting information (Supporting information (Appendix A)) in which the obtained X-ray difratograms and magnetization curves are shown.
5
st
1 harmonic
300
400
500
600
700
3.3. Fluorescent measurements In this section the photophysical study of the nanoparticles conjugated with the fluorescent dye is described. Fig. 5 shows the height-normalized absorption spectrum of the Alexa Fluor1 660 and the labelled sample. As can be seen, the absorption spectra are almost identical and show the principal absorption band centred at 668 nm, indicating the presence of the dye in the nanoparticles. Furthermore, a weak band in the UV region of the spectrum, around 250 nm, could include iron oxides such as hematite, magnetite or maghemite [42]. Fig. 6 shows the height-normalized fluorescence spectra of the fraction with the highest content of nanoparticles with dye in suspension at two excitation wavelengths, 250 and 620 nm. On the one hand, when the excitation of the sample takes place directly to the absorption band of the dye (620 nm, see Fig. 5) the emission band is obtained at 696 nm, emission band typical of Alexa Fluor 6601 dye, indicating its presence in the particles. In order to compare the fluorescence efficiency of Alexa 660 dye in solution and anchored at the nanoparticles, the ratio between the fluorescence intensity and the absorbance of the sample at the excitation wavelength is analysed (Fig. S6). In this way and assuming a quantum yield of around 0.37 for Alexa 660 in aqueous solution [36], an estimated quantum yield of around 0.13 is obtained for the dye at the nanoparticles in suspension On the other hand, when the excitation wavelength was fixed at 250 nm (absorption attributed mainly to the iron oxides present in the nanoparticles) the obtained band at 390 nm 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.
Fig. 6. Height-normalized fluorescence spectra of iron nanoparticles in aqueous buffer suspension at excitation wavelengths of 250 and 620 nm.
Although the absorption and fluorescence spectroscopic techniques indicate the presence of fluorescence dye in the suspension of nanoparticles, to confirm the anchorage to the nanoparticles surface confocal fluorescence time resolved microscopy measurements were carried out. This technique allows the study of the fluorescent 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 fluorescence in single particle can be obtained (Fig. 8). So, by positioning the excitation laser (640 nm) in the centre of each nanoparticle, the fluorescence spectrum of the anchored dye nanoparticle is obtained (Fig. 8). In addition, the figure includes the spectrum of dye in solution measured at the same conditions. The maximum of fluorescence are 696 nm for dye and 687 nm 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. 9 shows the fluorescence 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 fluorescence life time t = 1.8 ns, while the conjugated nanoparticles presents a biexponencial behaviour with: life time t 1 0.1–0.5 ns y t 2 = 1.5–1.7 ns (Fig. 9). These values have been obtained after the analysis of, at least, 10 individual particles. The short half lifetime, around 0.1–0.6 ns can be attributed to the light scattered by the nanoparticle itself and the obtained long half life time (t 2 = 1.5–1.7) is attributed to anchored dye to nanoparticle surface.
Absorbance
Alexa Fluor 660 NP + Alexa Fluor 660
300
400
800
Wavelength (nm)
500
600
700
Wavelength (nm)
Fig. 5. Height-notmalized absorption spectra of Alexa Fluor 660 dye and iron labeled nanoparticles in aqueous buffer suspension.
Fig. 7. Fluorescence microscopy image of single particles.
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fluorescent properties which could make possible to monitor the course of in vitro or in vivo samples using fluorescent microscopy red under excitation. The magnetic properties of synthesized nanoparticles added to its fluorescent response result in a suitable material for be detected by both magnetic and fluorescent techniques for combined MR/ Optical imaging applications. Acknowledgements
Fig. 8. Fluorescence spectrum of a single particle (red curve) and a diluted dye solution (black curve) registered in time-resolved fluorescence confocal microscope at excitation wavelength of 640 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Authors thank the Basque Country Government for financial support (ACTIMAT project, ETORTEK programme IE10-272) (Ayudas para apoyar las actividades de los grupos de investigación del sistema universitario vasco, IT718-13 and 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 http://dx.doi.org/10.1016/j. jphotochem.2015.09.004. References
Fig. 9. Fluorescence decay curves of Alexa Fluor 660 and two different regions.
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 fluorescence microscopy confirmed that the dye is labelled onto nanoparticles and maintains its fluorescent properties. Therefore, the trajectory of these nanoparticles may be monitored by fluorescence 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 10 and 15 nm. It is noteworthy that the synthesized nanoparticles are mainly zerovalent iron. The FemPEG nanoparticles were successfully functionalized and conjugated with a fluorescent dye. Thus, amine-reactive N-hydroxysuccinimidyl ester of Alexa Fluor 660 dye was conjugated to the nanoparticle surface. This dye produces bright far red fluorescence emission with a peak at 690 nm under red excitation light (in the clinic window). Studies of confocal fluorescence microscopy confirmed that the fluorescent dye is attached to the nanoparticles and retains its
[1] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Quantum dot bioconjugates for imaging, labelling and sensing, Nat. Mater. 4 (2005) 435– 446. http://dx.doi.org/10.1038/nmat1390. [2] X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, et al., Quantum dots for live cells, in vivo imaging, and diagnostics, Science 307 (80) (2005) 538–544, doi:http://dx.doi.org/10.1126/science.1104274. [3] K.L. Kelly, E. Coronado, L.L. Zhao, G.C. Schatz, The optical propierties of metal nanoparticles: the influence of size, shape, and dielectric environment, J. Phys. Chem. B 107 (2003) 668–677, doi:http://dx.doi.org/10.1021/jp026731y. [4] K. Woo, J. Hong, S. Choi, H.-W. Lee, J. Ahn, C.S. Kim, et al., Easy synthesis and magnetic properties of iron oxide nanoparticles, Chem. Mater. 16 (2004) 2814– 2818, doi:http://dx.doi.org/10.1021/cm049552x. [5] W.S. Seo, H.H. Jo, K. Lee, B. Kim, S.J. Oh, J.T. Park, Size-dependent magnetic properties of colloidal Mn3O4 and MnO nanoparticles, Angew. Chem. Int. Ed. 43 (2004) 1115–1117, doi:http://dx.doi.org/10.1002/anie.200352400. [6] M. Colombo, S. Carregal-Romero, M.F. Casula, L. Gutierrez, M.P. Morales, I.B. Bohm, et al., Biological applications of magnetic nanoparticles, Chem. Soc. Rev. 41 (2012) 4306–4334, doi:http://dx.doi.org/10.1039/c2cs15337h. [7] S. Cheong, P. Ferguson, K.W. Feindel, I.F. Hermans, P.T. Callaghan, C. Meyer, et al., Simple Synthesis and functionalization of iron nanoparticles for magnetic resonance imaging, Angew. Chem. Int. Ed. 50 (2011) 4206–4209, doi: http://dx.doi.org/10.1002/anie.201100562. [8] Q. Pankhurst, N. Thanh, S. Jones, J. Dobson, Progress in applications of magnetic nanoparticles in biomedicine, J. Phys. D. Appl. Phys. 42 (2009) , doi:http://dx. doi.org/10.1088/0022-3727/42/22/224001. [9] A.B. Salunkhe, V.M. Khot, S.H. Pawar, Magnetic hyperthermia with magnetic nanoparticles: a status review, Curr. Top. Med. Chem. 14 (2014) 572–594, doi: http://dx.doi.org/10.2174/1568026614666140118203550. [10] S. Peng, C. Wang, J. Xie, S. Sun, Synthesis and stabilization of monodisperse Fe nanoparticles, J. Am. Chem. Soc. 128 (2006) 10676–10677, doi:http://dx.doi. org/10.1021/ja063969h. [11] Z. Wang, X. Li, M. Gao, X. Zeng, One-step preparation of amorphous iron nanoparticles by laser ablation, Powder Technol. 215–216 (2012) 147–150, doi: http://dx.doi.org/10.1016/j.powtec.2011.09.039. [12] D. Farrell, S. Majetich, J. Wilcoxon, Preparation and characterization of monodisperse Fe nanoparticles, J. Phys. Chem. B. 107 (2003) 11022–11030, doi: http://dx.doi.org/10.1021/jp0351831. [13] C.S. Tiwary, S. Kashyap, K. Biswas, K. Chattopadhyay, Synthesis of pure iron magnetic nanoparticles in large quantity, J. Phys. D. Appl. Phys. 46 (2013) 385001, doi:http://dx.doi.org/10.1088/0022-3727/46/38/385001. [14] O. Veiseh, J. Gunn, M. Zhang, Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging, Adv. Drug Deliv. Rev. 62 (2011) 284– 304, doi:http://dx.doi.org/10.1016/j.addr.2009.11.002 Design. [15] J.E. Macdonald, J. a Kelly, J.G.C. Veinot, Iron/iron oxide nanoparticle sequestration of catalytic metal impurities from aqueous media and organic reaction products, Langmuir 23 (2007) 9543–9545, doi:http://dx.doi.org/ 10.1021/la7011827. [16] I. San Román, M.L. Alonso, L. Bartolomé, E.G. Galdames, M.O. oiti, et al., Relevance study of bare and coated zero valent iron nanoparticles for lindane degradation from its by-product monitorization, Chemosphere 93 (2013) 1324–1332, doi:http://dx.doi.org/10.1016/j.chemosphere.2013.07.050.
N. Pérez et al. / Journal of Photochemistry and Photobiology A: Chemistry 315 (2016) 1–7 [17] I. Banerjee, R.C. Pangule, R.S. Kane, Antifouling coatings: recent developments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms, Adv. Mater. 23 (2011) 690–718, doi:http://dx.doi.org/10.1002/ adma.201001215. [18] S. Chen, L. Li, C. Zhao, J. Zheng, Surface hydration: principles and applications toward low-fouling/nonfouling biomaterials, Polymer (Guildf.) 51 (2010) 5283–5293, doi:http://dx.doi.org/10.1016/j.polymer.2010.08.022. [19] J. Jokerst V, T. Lobovkina, R.N. Zare, S.S. Gambhir, Nanoparticle PEGylation for imaging and therapy, Nanomedicine (Lond.) 6 (2011) 715–728, doi:http://dx. doi.org/10.2217/nnm.11.19. [20] Q. Wang, Y. Bao, J. Ahire, Y. Chao, Co-encapsulation of biodegradable nanoparticles with silicon quantum dots and quercetin for monitored delivery, Adv. Healthcare Mater. 2 (2013) 459–466, doi:http://dx.doi.org/10.1002/ adhm.201200178. [21] M. Tobı’o, A. Sánchez, A. Vila, I. Soriano, C. Evora, J. Vila-Jato, et al., The role of PEG on the stability in digestive fluids and in vivo fate of PEG-PLA nanoparticles following oral administration, Colloids Surf. B Biointerfaces 18 (2000) 315–323, doi:http://dx.doi.org/10.1016/s0927-7765(99)00157-5. [22] A. Ruiz, G. Salas, M. Calero, Y. Hernández, A. Villanueva, F. Herranz, et al., Shortchain PEG molecules strongly bound to magnetic nanoparticle for MRI long circulating agents, Acta Biomater. 9 (2013) 6421–6430, doi:http://dx.doi.org/ 10.1016/j.actbio.2012.12.032. [23] A. Mukhopadhyay, N. Joshi, K. Chattopadhyay, G. De, A facile synthesis of PEGcoated magnetite (Fe3O4) nanoparticles and their prevention of the reduction of cytochrome C, ACS Appl. Mater. Interfaces 4 (2011) 142–149, doi:http://dx. doi.org/10.1021/am201166m. [24] A.S. Karakoti, S. Das, S. Thevuthasan, S. Seal, PEGylated inorganic nanoparticles, Angew. Chem. Int. Ed. 50 (2011) 1980–1994, doi:http://dx.doi. org/10.1002/anie.201002969. [25] H. Otsuka, Y. Nagasaki, K. Kataoka, PEGylated nanoparticles for biological and pharmaceutical applications, Adv. Drug Deliv. Rev. 64 (2012) 246–255, doi: http://dx.doi.org/10.1016/j.addr.2012.09.022. [26] K.G. Neoh, E.T. Kang, Functionalization of inorganic nanoparticles with polymers for stealth biomedical applications, Polym. Chem. 2 (2011) 747, doi: http://dx.doi.org/10.1039/c0py00266f. [27] S. Mao, M. Neu, O. Germershaus, O. Merkel, J. Sitterberg, U. Bakowsky, et al., Influence of Polyethylene Glycol Chain Length on the Physicochemical and Biological Properties of Poly (ethylene imine)-graft-Poly (ethylene glycol) Block Copolymer/SiRNA Polyplexes, (2006). [28] L. Zhou, J. Yuan, Y. Wei, Core-shell structural iron oxide hybrid nanoparticles: from controlled synthesis to biomedical applications, J. Mater. Chem. 21 (2011) 2823, doi:http://dx.doi.org/10.1039/c0jm02172e. [29] H. Xu, Z.P. Aguilar, L. Yang, M. Kuang, H. Duan, Y. Xiong, et al., Antibody conjugated magnetic iron oxide nanoparticles for cancer cell separation in fresh whole blood, Biomaterials 32 (2011) 9758–9765, doi:http://dx.doi.org/ 10.1016/j.biomaterials.2011.08.076. [30] K. Cheng, D. Shen, M.T. Hensley, R. Middleton, B. Sun, W. Liu, et al., Magnetic antibody-linked nanomatchmakers for therapeutic cell targeting, Nat. Commun. 5 (2014) 4880, doi:http://dx.doi.org/10.1038/ncomms5880.
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[31] N. Chekina, D. Horák, P. Jendelová, M. Trchová, M.J. Beneš, M. Hrubý, et al., Fluorescent magnetic nanoparticles for biomedical applications, J. Mater. Chem. 21 (2011) 7630, doi:http://dx.doi.org/10.1039/c1jm10621j. [32] X. Xiong, F. Song, G. Chen, W. Sun, J. Wang, P. Gao, et al., Construction of longwavelength fluorescein analogues and their application as fluorescent probes, Chem. A Eur. J. 19 (2013) 6538–6545, doi:http://dx.doi.org/10.1002/ chem.201300418. [33] L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He, et al., A unique approach to development of near-infrared fluorescent sensors for in vivo imaging, J. Am. Chem. Soc. 134 (2012) 13510–13523, doi:http://dx.doi.org/10.1021/ja305802v. czewski, J.L. Lakshmi, S. Bin Dolmanan, S. Tripathy, V.H.B. Ho, [34] S.K. Yen, D. Jan et al., Design and synthesis of polymer-functionalized NIR fluorescent dyesmagnetic nanoparticles for bioimaging, ACS Nano 7 (2013) 6796–6805, doi: http://dx.doi.org/10.1021/nn401734t. [35] L. Josephson, M.F. Kircher, U. Mahmood, Y. Tang, R. Weissleder, Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes, Bioconjugate Chem. 13 (2002) 554–560, doi:http://dx.doi.org/10.1021/ bc015555d. [36] J. Li, L. Zheng, H. Cai, W. Sun, M. Shen, G. Zhang, et al., Facile one-pot synthesis of Fe3O4@Au composite nanoparticles for dual-mode MR/CT imaging applications, ACS Appl. Mater. Interfaces 5 (2013) 10357–10366, doi:http://dx. doi.org/10.1021/am4034526. [37] W. He, L. Cheng, L. Zhang, X. Jiang, Z. Liu, Z. Cheng, et al., Bifunctional nanoparticles with magnetism and NIR fluorescence: controlled synthesis from combination of AGET ATRP and click reaction, Nanotechnology 25 (2014) 045602, doi:http://dx.doi.org/10.1088/0957-4484/25/4/045602. [38] J.M. Harris, E.C. Struck, M.G. Case, M.S. Paley, M. Yalpani, J.M. Van Alstine, et al., Synthesis and characterization of poly(ethylene glycol) derivatives, J. Polym. Sci. Polym. Chem. Ed. 22 (1984) 341–352, doi:http://dx.doi.org/10.1002/ pol.1984.170220207. [39] 1–4. Molecular Probes Invitrogen detection technologies, Alexa Fluor1660 Protein Labeling Kit, No Title, (n.d.). [40] D. Ortega, N. Pérez, J.L. Vilas, J.S. Garitaonandia, K. Suzuki, J.R. Marín, et al., Nonylphenol polyethoxylate coated body-center-cubic iron nanocrystals for ferrofluids with technical applications, J. Appl. Phys. 113 (2013) 2013–2016, doi:http://dx.doi.org/10.1063/1.4794880. [41] Y.-P. Sun, X. Li, J. Cao, W. Zhang, H.P. Wang, Characterization of zero-valent iron nanoparticles, Adv. Colloid Interface Sci. 120 (2006) 47–56, doi:http://dx.doi. org/10.1016/j.cis.2006.03.001. [42] R.B. Cao, X.Q. Chen, W.H. Shen, Z. Long, A facile route to synthesize nanohematite colloid, Mater. Lett. 65 (2011) 3298–3300, doi:http://dx.doi.org/ 10.1016/j.matlet.2011.03.006. [43] M.B.J. Roeffaers, G. De Cremer, H. Uji-i, B. Muls, B.F. Sels, P. a Jacobs, et al., Single-molecule fluorescence spectroscopy in (bio) catalysis, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 12603–12609, doi:http://dx.doi.org/10.1073/ pnas.0610755104.
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
Synthesis and characterization of near-infrared fluorescent and magnetic iron zero-valent nanoparticles Nagore Péreza , Leire Ruiz-Rubioa,* , José Luis Vilasa,b , Matilde Rodrígueza , Virginia Martinez-Martineza , Luis M. Leóna,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
A B S T R A C T
Article history: Received 4 May 2015 Received in revised form 4 September 2015 Accepted 6 September 2015 Available online 9 September 2015
Polyethylene glycol coated iron nanoparticles were synthesized by a microemulsion method, modified 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 fluorescent dye, Alexa Fluor 660, confirmed that the fluorescent dye is attached to the nanoparticles and retains its fluorescent 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 fluorescent properties being of particular importance for potential applications in bioscience. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Iron zero valent nanoparticles Fluorescent Magnetic Polyethylenglycol
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 (Fe3O4) and maghemite (g-Fe2O3). Elemental iron has a significantly 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 (218 emu g 1 in bulk), and a Curie temperature which is high enough for the majority of practical applications. However, obtaining Fe nanoparticles, relatively free of oxide (usually Fe3O4), is still a challenge, to a large extent, not overcome [10–13].
* Corresponding author. E-mail address: [email protected] (L. Ruiz-Rubio). http://dx.doi.org/10.1016/j.jphotochem.2015.09.004 1010-6030/ ã 2015 Elsevier B.V. All rights reserved.
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-specific 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 nanoparticles 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 significant applications in the structure stabilization [26–28]. Finally, the polymeric coatings of the nanoparticles could be conjugated with antibodies or fluorescent dyes adding different
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properties to the system [29–31]. That is, fluorescent-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 systems. Compared with fluorescent 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 autofluorescence 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 specific difficulty in the preparation of fluorescent magnetic nanoparticles due to the risk of quenching of the fluorophore 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 fluorescent molecule or by attaching an appropriate spacer to the fluorophore. Most fluorescent magnetic nanoparticles thus have a core-shell structure. Several studies have been devoted to develop iron oxide nanoparticles conjugated with fluorescent dyes, in order to obtain dual-responsive nanoparticles, with magnetic and fluorescent 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 significant lack on studies about iron nanoparticles functionalized with fluorophores [37]. The aim of this work is to synthesize iron nanoparticles coated with a PEG-derivative and functionalized with a fluorescent 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 fluorescent 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.
systems (sample FePEG-02). The procedure followed in the first case is described here. A surfactant solution prepared by dissolving 31.5 g of polyethylene glycol in 105 mL of cyclohexane was maintained under stirring and degassed for 10 min under N2 atmosphere. Next, 6 mL of 0.33 M FeCl24H2O were added to the surfactant solution, stirred and degassed for 10 min. Metal particles were formed inside the reverse micelles via reduction of the metal salt using an excess of NaBH4 (6 mL, 1.76 M). After a few minutes, the reaction was quenched by adding 50 mL of chloroform and 50 mL 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 flask reaction, will result the second micellar system. Definitely, 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. Specifically, 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, 0.40 g of iron salt, 0.20 g of the reducing agent, 105 cm3 of cyclohexane and 6.0 g of water. The concentration of surfactant in this system was 0.095 M.
2. Materials and methods
2.3.1. Modification of mPEG Polyethylene glycol methyl ether (mPEG) of molecular weight 2000 g mol 1 was firstly 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.
2.1. Chemicals All chemicals were reagent grade and used without purification. Ferrous chloride tetrahydrate (FeCl24H2O), 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) of 1000 g mol 1 molecular weight and methoxy polyethylene glycol (mPEG) of 2000 g mol 1 molecular weight were obtained from Sigma– Aldrich. Deionized Millipore Milli-Q water was used in all experiments. Alexa Fluor1660 Protein 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
2.3. Functionalization of nanoparticles and labelling with fluorescent dye The incorporation of the fluorescent molecule to the nanoparticles consists of several steps. Firstly, functionalized nanoparticles are synthesized and then the fluorophore is anchored. After that the labelled nanoparticles must be purified to take out the excess dye by size-exclusion chromatography.
2.3.2. Synthesis of nanoparticles with mPEG-NH2 and PEG The synthesis of nanoparticles was performed by the method previously described for one micellar system. Owing to the small amount of material fluorescent necessary, the appropriate amount of mPEG-NH2 was used, and the rest was PEG surfactant, as already shown, to provide adequate protection to the nanoparticles. The surfactant consisted of a mixture of 7.5 g of PEG and 217 mg of mPEG-NH2, amounts required to have a total surfactant concentration of 0.30 M. 2.3.3. Labelling of nanoparticles The interaction of metal nanoparticles with fluorophores near its surface affects the intensity of their emission being critical the distance between the fluorophore and the surface of the nanoparticle so that the fluorescence is quenched when the distance is too short. For this study Alexa Fluor 660 was used. This is a succinimidyl ester of Alexa Fluor which exhibits bright fluorescence and high photostability characteristics allowing us to
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capture images that were previously unattainable with conventional fluorophores. Moreover it provides an efficient and convenient way to selectively link to primary amines. On the other hand, its absorption and fluorescence bands are far from those of the nanoparticles, so that the spectral overlapping is negligible. The PEGylated nanoparticles were fluorescently labelled by reaction with Alexa Fluor 660 carboxylic acid succinimidyl ester which formed a chemical bond with the NH2 group of mPEG-NH2. For that, the procedure established by Invitrogen [39] was followed. Briefly, a solution of sodium bicarbonate was added to the nanoparticles suspension in order to reach a pH between 7,5 and 8,5 since succinimidyl esters react efficiently at this pH range. The reactive dye was added to the solution and the reaction mixture was stirred for 1 h at room temperature. Separation of the labelled nanoparticles from dye which has remained unreacted was carried out using a purification column containing the Bio-Rad BioGel P30 resin. 2.4. Characterization of nanoparticles The crystallite phase of the coated nanoparticles was identified 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 200 equipment operating at an accelerating voltage of 200 KV. For this, a drop of dilute methanol solution of the nanoparticles was placed onto a copper grid coated with carbon film 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 Mössbauer 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 hyperfine field (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 200–900 nm. The fluorescence spectra were performed on a SPEX fluorimeter (Fluorolog 3-22). The emission spectra were recorded in the 250–800 nm range, by exciting at different wavelengths, depending on the sample. Fluorescence single-particle measurements were performed in a time-resolved fluorescence confocal microscope (model Micro
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Time 200, PicoQuant). Fluorescence lifetime images (FLIM) are processed with ShymPhotime software (Picoquant) by sorting all photons of one pixel into a histogram and fitted to an exponential decay function to extract lifetime information; the procedure was repeated for every pixel in the image. A 640 nm pulsed laser diode, with 70 ps pulses was used as excitation source. Spectra were recorded by directing the emission beam to an exit port, where a spectrograph (model Shamrock 300 mm) 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 measurements 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 (11 0), (2 0 0), and (2 11) families of planes of the bcc lattice reported for the a-Fe phase. The dimension of the crystallites, Dhkl, was estimated by Scherrer equation in 27.8 nm. The nanoparticles obtained with mPEG as surfactant present a diffractogram with a peak of high intensity at 2u = 45 , corresponding to the bcc lattice (Fig. 1b). This kind of diffractogram is characteristic of samples with low crystallinity and very polydisperse 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 3.8 nm and its own polymeric coating of about 6 nm. According to XRD results, the FemPEG-01 sample was very polydisperse and it was very difficult to obtain a mean diameter. In general, the size of the nanoparticles was between 10 and 20 nm. The values obtained are similar to those obtained when using nonylphenypentaethoxylated (NP5) [40] as surfactant whose value was around 10 nm (Fe core 7.5 nm and polymeric shell 2.8 nm). 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 field hysteresis loops were measured using VSM to assess the magnetic properties of the synthesized nanoparticles. The saturation magnetization values were normalized to the mass of nanoparticles to yield the specific magnetization, Ms (emu g 1).
Fig. 1. X-ray diffractograms of the synthesized iron nanoparticles: (a) Polyethylenglycol coated samples and (b) polyethylene glycol methyl ether coated sample.
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Fig. 2. Micrographs of (a) FePEG-02, (b) FePEG-04 and (c) FemPEG-01 samples.
Fig. 3 shows the magnetic hysteresis loops of the samples at room temperature. The saturation magnetization of FePEG nanoparticles is shown in Table 1. The saturation magnetization arises from both the iron core (218 emu g 1), and the iron oxide shell (for Fe3O4 80–92 emu g 1), 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 15 mT and a remanence less than 25 A m2 kg 1. This suggested that the particles could aggregate after the removal of the external field due to the remaining magnetization. 57 Fe Mössbauer spectroscopy measurements were carried out for the FePEG-04 sample due to it has the best magnetic properties
Fig. 3. Magnetization curves.
Table 1 Saturation magnetization (Ms), coercitive field (Hc) and remanent magnetization. Muestra
Ms (A m2 kg
FePEG-02 FePEG-04 FemPEG-01
116 135 108
1
)
Hc (mT)
Mr (A m2 kg
15.3 13.2 16.8
19.9 21.3 19.2
1
)
Fig. 4. RT Mössbauer spectrum for FePEG-04 sample.
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exc 250 exc 620
Fluorescence Intensity (a.u.)
of the studied samples (Fig. 4). The RT Mössbauer spectrum qualitatively consist in a sextet (62% of the total area), attributed to bcc Fe (BHF = 32.89 T and d = 0.106 mm s 1) 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 confirmed by the XPS (Apendix A, Fig. S5) where the peaks at 710.30, 718.98 (small peak) and 723.32 eV represent the binding energies of Fe (2p3/2) shake-up satellite 2p3/2 and 2p1/2, respectively. In addition, a small shoulder at 705,87 eV suggest the peak of 2p3/2 of zero-valent iron [41]. All the studied systems present a high reproducibility as could be confirm in the supporting information (Supporting information (Appendix A)) in which the obtained X-ray difratograms and magnetization curves are shown.
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600
700
3.3. Fluorescent measurements In this section the photophysical study of the nanoparticles conjugated with the fluorescent dye is described. Fig. 5 shows the height-normalized absorption spectrum of the Alexa Fluor1 660 and the labelled sample. As can be seen, the absorption spectra are almost identical and show the principal absorption band centred at 668 nm, indicating the presence of the dye in the nanoparticles. Furthermore, a weak band in the UV region of the spectrum, around 250 nm, could include iron oxides such as hematite, magnetite or maghemite [42]. Fig. 6 shows the height-normalized fluorescence spectra of the fraction with the highest content of nanoparticles with dye in suspension at two excitation wavelengths, 250 and 620 nm. On the one hand, when the excitation of the sample takes place directly to the absorption band of the dye (620 nm, see Fig. 5) the emission band is obtained at 696 nm, emission band typical of Alexa Fluor 6601 dye, indicating its presence in the particles. In order to compare the fluorescence efficiency of Alexa 660 dye in solution and anchored at the nanoparticles, the ratio between the fluorescence intensity and the absorbance of the sample at the excitation wavelength is analysed (Fig. S6). In this way and assuming a quantum yield of around 0.37 for Alexa 660 in aqueous solution [36], an estimated quantum yield of around 0.13 is obtained for the dye at the nanoparticles in suspension On the other hand, when the excitation wavelength was fixed at 250 nm (absorption attributed mainly to the iron oxides present in the nanoparticles) the obtained band at 390 nm 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.
Fig. 6. Height-normalized fluorescence spectra of iron nanoparticles in aqueous buffer suspension at excitation wavelengths of 250 and 620 nm.
Although the absorption and fluorescence spectroscopic techniques indicate the presence of fluorescence dye in the suspension of nanoparticles, to confirm the anchorage to the nanoparticles surface confocal fluorescence time resolved microscopy measurements were carried out. This technique allows the study of the fluorescent 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 fluorescence in single particle can be obtained (Fig. 8). So, by positioning the excitation laser (640 nm) in the centre of each nanoparticle, the fluorescence spectrum of the anchored dye nanoparticle is obtained (Fig. 8). In addition, the figure includes the spectrum of dye in solution measured at the same conditions. The maximum of fluorescence are 696 nm for dye and 687 nm 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. 9 shows the fluorescence 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 fluorescence life time t = 1.8 ns, while the conjugated nanoparticles presents a biexponencial behaviour with: life time t 1 0.1–0.5 ns y t 2 = 1.5–1.7 ns (Fig. 9). These values have been obtained after the analysis of, at least, 10 individual particles. The short half lifetime, around 0.1–0.6 ns can be attributed to the light scattered by the nanoparticle itself and the obtained long half life time (t 2 = 1.5–1.7) is attributed to anchored dye to nanoparticle surface.
Absorbance
Alexa Fluor 660 NP + Alexa Fluor 660
300
400
800
Wavelength (nm)
500
600
700
Wavelength (nm)
Fig. 5. Height-notmalized absorption spectra of Alexa Fluor 660 dye and iron labeled nanoparticles in aqueous buffer suspension.
Fig. 7. Fluorescence microscopy image of single particles.
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fluorescent properties which could make possible to monitor the course of in vitro or in vivo samples using fluorescent microscopy red under excitation. The magnetic properties of synthesized nanoparticles added to its fluorescent response result in a suitable material for be detected by both magnetic and fluorescent techniques for combined MR/ Optical imaging applications. Acknowledgements
Fig. 8. Fluorescence spectrum of a single particle (red curve) and a diluted dye solution (black curve) registered in time-resolved fluorescence confocal microscope at excitation wavelength of 640 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Authors thank the Basque Country Government for financial support (ACTIMAT project, ETORTEK programme IE10-272) (Ayudas para apoyar las actividades de los grupos de investigación del sistema universitario vasco, IT718-13 and 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 http://dx.doi.org/10.1016/j. jphotochem.2015.09.004. References
Fig. 9. Fluorescence decay curves of Alexa Fluor 660 and two different regions.
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 fluorescence microscopy confirmed that the dye is labelled onto nanoparticles and maintains its fluorescent properties. Therefore, the trajectory of these nanoparticles may be monitored by fluorescence 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 10 and 15 nm. It is noteworthy that the synthesized nanoparticles are mainly zerovalent iron. The FemPEG nanoparticles were successfully functionalized and conjugated with a fluorescent dye. Thus, amine-reactive N-hydroxysuccinimidyl ester of Alexa Fluor 660 dye was conjugated to the nanoparticle surface. This dye produces bright far red fluorescence emission with a peak at 690 nm under red excitation light (in the clinic window). Studies of confocal fluorescence microscopy confirmed that the fluorescent dye is attached to the nanoparticles and retains its
[1] I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Quantum dot bioconjugates for imaging, labelling and sensing, Nat. Mater. 4 (2005) 435– 446. http://dx.doi.org/10.1038/nmat1390. [2] X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, et al., Quantum dots for live cells, in vivo imaging, and diagnostics, Science 307 (80) (2005) 538–544, doi:http://dx.doi.org/10.1126/science.1104274. [3] K.L. Kelly, E. Coronado, L.L. Zhao, G.C. Schatz, The optical propierties of metal nanoparticles: the influence of size, shape, and dielectric environment, J. Phys. Chem. B 107 (2003) 668–677, doi:http://dx.doi.org/10.1021/jp026731y. [4] K. Woo, J. Hong, S. Choi, H.-W. Lee, J. Ahn, C.S. Kim, et al., Easy synthesis and magnetic properties of iron oxide nanoparticles, Chem. Mater. 16 (2004) 2814– 2818, doi:http://dx.doi.org/10.1021/cm049552x. [5] W.S. Seo, H.H. Jo, K. Lee, B. Kim, S.J. Oh, J.T. Park, Size-dependent magnetic properties of colloidal Mn3O4 and MnO nanoparticles, Angew. Chem. Int. Ed. 43 (2004) 1115–1117, doi:http://dx.doi.org/10.1002/anie.200352400. [6] M. Colombo, S. Carregal-Romero, M.F. Casula, L. Gutierrez, M.P. Morales, I.B. Bohm, et al., Biological applications of magnetic nanoparticles, Chem. Soc. Rev. 41 (2012) 4306–4334, doi:http://dx.doi.org/10.1039/c2cs15337h. [7] S. Cheong, P. Ferguson, K.W. Feindel, I.F. Hermans, P.T. Callaghan, C. Meyer, et al., Simple Synthesis and functionalization of iron nanoparticles for magnetic resonance imaging, Angew. Chem. Int. Ed. 50 (2011) 4206–4209, doi: http://dx.doi.org/10.1002/anie.201100562. [8] Q. Pankhurst, N. Thanh, S. Jones, J. Dobson, Progress in applications of magnetic nanoparticles in biomedicine, J. Phys. D. Appl. Phys. 42 (2009) , doi:http://dx. doi.org/10.1088/0022-3727/42/22/224001. [9] A.B. Salunkhe, V.M. Khot, S.H. Pawar, Magnetic hyperthermia with magnetic nanoparticles: a status review, Curr. Top. Med. Chem. 14 (2014) 572–594, doi: http://dx.doi.org/10.2174/1568026614666140118203550. [10] S. Peng, C. Wang, J. Xie, S. Sun, Synthesis and stabilization of monodisperse Fe nanoparticles, J. Am. Chem. Soc. 128 (2006) 10676–10677, doi:http://dx.doi. org/10.1021/ja063969h. [11] Z. Wang, X. Li, M. Gao, X. Zeng, One-step preparation of amorphous iron nanoparticles by laser ablation, Powder Technol. 215–216 (2012) 147–150, doi: http://dx.doi.org/10.1016/j.powtec.2011.09.039. [12] D. Farrell, S. Majetich, J. Wilcoxon, Preparation and characterization of monodisperse Fe nanoparticles, J. Phys. Chem. B. 107 (2003) 11022–11030, doi: http://dx.doi.org/10.1021/jp0351831. [13] C.S. Tiwary, S. Kashyap, K. Biswas, K. Chattopadhyay, Synthesis of pure iron magnetic nanoparticles in large quantity, J. Phys. D. Appl. Phys. 46 (2013) 385001, doi:http://dx.doi.org/10.1088/0022-3727/46/38/385001. [14] O. Veiseh, J. Gunn, M. Zhang, Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging, Adv. Drug Deliv. Rev. 62 (2011) 284– 304, doi:http://dx.doi.org/10.1016/j.addr.2009.11.002 Design. [15] J.E. Macdonald, J. a Kelly, J.G.C. Veinot, Iron/iron oxide nanoparticle sequestration of catalytic metal impurities from aqueous media and organic reaction products, Langmuir 23 (2007) 9543–9545, doi:http://dx.doi.org/ 10.1021/la7011827. [16] I. San Román, M.L. Alonso, L. Bartolomé, E.G. Galdames, M.O. oiti, et al., Relevance study of bare and coated zero valent iron nanoparticles for lindane degradation from its by-product monitorization, Chemosphere 93 (2013) 1324–1332, doi:http://dx.doi.org/10.1016/j.chemosphere.2013.07.050.
N. Pérez et al. / Journal of Photochemistry and Photobiology A: Chemistry 315 (2016) 1–7 [17] I. Banerjee, R.C. Pangule, R.S. Kane, Antifouling coatings: recent developments in the design of surfaces that prevent fouling by proteins, bacteria, and marine organisms, Adv. Mater. 23 (2011) 690–718, doi:http://dx.doi.org/10.1002/ adma.201001215. [18] S. Chen, L. Li, C. Zhao, J. Zheng, Surface hydration: principles and applications toward low-fouling/nonfouling biomaterials, Polymer (Guildf.) 51 (2010) 5283–5293, doi:http://dx.doi.org/10.1016/j.polymer.2010.08.022. [19] J. Jokerst V, T. Lobovkina, R.N. Zare, S.S. Gambhir, Nanoparticle PEGylation for imaging and therapy, Nanomedicine (Lond.) 6 (2011) 715–728, doi:http://dx. doi.org/10.2217/nnm.11.19. [20] Q. Wang, Y. Bao, J. Ahire, Y. Chao, Co-encapsulation of biodegradable nanoparticles with silicon quantum dots and quercetin for monitored delivery, Adv. Healthcare Mater. 2 (2013) 459–466, doi:http://dx.doi.org/10.1002/ adhm.201200178. [21] M. Tobı’o, A. Sánchez, A. Vila, I. Soriano, C. Evora, J. Vila-Jato, et al., The role of PEG on the stability in digestive fluids and in vivo fate of PEG-PLA nanoparticles following oral administration, Colloids Surf. B Biointerfaces 18 (2000) 315–323, doi:http://dx.doi.org/10.1016/s0927-7765(99)00157-5. [22] A. Ruiz, G. Salas, M. Calero, Y. Hernández, A. Villanueva, F. Herranz, et al., Shortchain PEG molecules strongly bound to magnetic nanoparticle for MRI long circulating agents, Acta Biomater. 9 (2013) 6421–6430, doi:http://dx.doi.org/ 10.1016/j.actbio.2012.12.032. [23] A. Mukhopadhyay, N. Joshi, K. Chattopadhyay, G. De, A facile synthesis of PEGcoated magnetite (Fe3O4) nanoparticles and their prevention of the reduction of cytochrome C, ACS Appl. Mater. Interfaces 4 (2011) 142–149, doi:http://dx. doi.org/10.1021/am201166m. [24] A.S. Karakoti, S. Das, S. Thevuthasan, S. Seal, PEGylated inorganic nanoparticles, Angew. Chem. Int. Ed. 50 (2011) 1980–1994, doi:http://dx.doi. org/10.1002/anie.201002969. [25] H. Otsuka, Y. Nagasaki, K. Kataoka, PEGylated nanoparticles for biological and pharmaceutical applications, Adv. Drug Deliv. Rev. 64 (2012) 246–255, doi: http://dx.doi.org/10.1016/j.addr.2012.09.022. [26] K.G. Neoh, E.T. Kang, Functionalization of inorganic nanoparticles with polymers for stealth biomedical applications, Polym. Chem. 2 (2011) 747, doi: http://dx.doi.org/10.1039/c0py00266f. [27] S. Mao, M. Neu, O. Germershaus, O. Merkel, J. Sitterberg, U. Bakowsky, et al., Influence of Polyethylene Glycol Chain Length on the Physicochemical and Biological Properties of Poly (ethylene imine)-graft-Poly (ethylene glycol) Block Copolymer/SiRNA Polyplexes, (2006). [28] L. Zhou, J. Yuan, Y. Wei, Core-shell structural iron oxide hybrid nanoparticles: from controlled synthesis to biomedical applications, J. Mater. Chem. 21 (2011) 2823, doi:http://dx.doi.org/10.1039/c0jm02172e. [29] H. Xu, Z.P. Aguilar, L. Yang, M. Kuang, H. Duan, Y. Xiong, et al., Antibody conjugated magnetic iron oxide nanoparticles for cancer cell separation in fresh whole blood, Biomaterials 32 (2011) 9758–9765, doi:http://dx.doi.org/ 10.1016/j.biomaterials.2011.08.076. [30] K. Cheng, D. Shen, M.T. Hensley, R. Middleton, B. Sun, W. Liu, et al., Magnetic antibody-linked nanomatchmakers for therapeutic cell targeting, Nat. Commun. 5 (2014) 4880, doi:http://dx.doi.org/10.1038/ncomms5880.
7
[31] N. Chekina, D. Horák, P. Jendelová, M. Trchová, M.J. Beneš, M. Hrubý, et al., Fluorescent magnetic nanoparticles for biomedical applications, J. Mater. Chem. 21 (2011) 7630, doi:http://dx.doi.org/10.1039/c1jm10621j. [32] X. Xiong, F. Song, G. Chen, W. Sun, J. Wang, P. Gao, et al., Construction of longwavelength fluorescein analogues and their application as fluorescent probes, Chem. A Eur. J. 19 (2013) 6538–6545, doi:http://dx.doi.org/10.1002/ chem.201300418. [33] L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He, et al., A unique approach to development of near-infrared fluorescent sensors for in vivo imaging, J. Am. Chem. Soc. 134 (2012) 13510–13523, doi:http://dx.doi.org/10.1021/ja305802v. czewski, J.L. Lakshmi, S. Bin Dolmanan, S. Tripathy, V.H.B. Ho, [34] S.K. Yen, D. Jan et al., Design and synthesis of polymer-functionalized NIR fluorescent dyesmagnetic nanoparticles for bioimaging, ACS Nano 7 (2013) 6796–6805, doi: http://dx.doi.org/10.1021/nn401734t. [35] L. Josephson, M.F. Kircher, U. Mahmood, Y. Tang, R. Weissleder, Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes, Bioconjugate Chem. 13 (2002) 554–560, doi:http://dx.doi.org/10.1021/ bc015555d. [36] J. Li, L. Zheng, H. Cai, W. Sun, M. Shen, G. Zhang, et al., Facile one-pot synthesis of Fe3O4@Au composite nanoparticles for dual-mode MR/CT imaging applications, ACS Appl. Mater. Interfaces 5 (2013) 10357–10366, doi:http://dx. doi.org/10.1021/am4034526. [37] W. He, L. Cheng, L. Zhang, X. Jiang, Z. Liu, Z. Cheng, et al., Bifunctional nanoparticles with magnetism and NIR fluorescence: controlled synthesis from combination of AGET ATRP and click reaction, Nanotechnology 25 (2014) 045602, doi:http://dx.doi.org/10.1088/0957-4484/25/4/045602. [38] J.M. Harris, E.C. Struck, M.G. Case, M.S. Paley, M. Yalpani, J.M. Van Alstine, et al., Synthesis and characterization of poly(ethylene glycol) derivatives, J. Polym. Sci. Polym. Chem. Ed. 22 (1984) 341–352, doi:http://dx.doi.org/10.1002/ pol.1984.170220207. [39] 1–4. Molecular Probes Invitrogen detection technologies, Alexa Fluor1660 Protein Labeling Kit, No Title, (n.d.). [40] D. Ortega, N. Pérez, J.L. Vilas, J.S. Garitaonandia, K. Suzuki, J.R. Marín, et al., Nonylphenol polyethoxylate coated body-center-cubic iron nanocrystals for ferrofluids with technical applications, J. Appl. Phys. 113 (2013) 2013–2016, doi:http://dx.doi.org/10.1063/1.4794880. [41] Y.-P. Sun, X. Li, J. Cao, W. Zhang, H.P. Wang, Characterization of zero-valent iron nanoparticles, Adv. Colloid Interface Sci. 120 (2006) 47–56, doi:http://dx.doi. org/10.1016/j.cis.2006.03.001. [42] R.B. Cao, X.Q. Chen, W.H. Shen, Z. Long, A facile route to synthesize nanohematite colloid, Mater. Lett. 65 (2011) 3298–3300, doi:http://dx.doi.org/ 10.1016/j.matlet.2011.03.006. [43] M.B.J. Roeffaers, G. De Cremer, H. Uji-i, B. Muls, B.F. Sels, P. a Jacobs, et al., Single-molecule fluorescence spectroscopy in (bio) catalysis, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 12603–12609, doi:http://dx.doi.org/10.1073/ pnas.0610755104.