Synthesis and characterization of functionalized core–shell γFe2O3–SiO2 nanoparticles

ARTICLE IN PRESS Journal of Magnetism and Magnetic Materials 321 (2009) 1408–1413

Contents lists available at ScienceDirect

Journal of Magnetism and Magnetic Materials journal homepage: www.elsevier.com/locate/jmmm

Synthesis and characterization of functionalized core–shell gFe2O3–SiO2 nanoparticles Vincent Maurice, Thomas Georgelin, Jean-Michel Siaugue , Vale´rie Cabuil Laboratoire Liquides Ioniques et Interfaces Charge´es (LI2C), Universite´ Pierre et Marie Curie (Paris 6), UMR UPMC/CNRS/ESPCI 7612, Case 51, 4 place Jussieu, 75252 Paris Cedex 05, France

a r t i c l e in f o

a b s t r a c t

Available online 20 February 2009

Amino and/or polyethyleneglycol (PEG) functionalized core–shell gFe2O3–SiO2 magnetic nanoparticles were synthesized and characterized. Amino–PEG-functionalized core–shell nanoparticles have calibrated sizes and a good colloidal stability. These bi-functionalized core–shell nanoparticles are potentially useful as biocompatible particles for magnetically targeted chemotherapy. & 2009 Elsevier B.V. All rights reserved.

Keywords: Magnetic nanoparticle Silica core–shell Bi-functionalization Biomedical application

1. Introduction Past decades have shown a great improvement of magnetic nanoparticles for applications in biomedicine or in biology [1–5]. These materials are principally used as contrast agents [6], for drug delivery [7], hyperthermia [8], cell sorting and biomolecule transport [9]. Nanometer-sized particles have a large specific surface area allowing to anchor a high number of biomolecules. Moreover, for in vivo applications, their nanosize range of materials ranks them at a dimension comparable to a protein or a virus and this small size improves tissue diffusion. Lastly, magnetic properties of nanoparticles allow for biomolecule transport under an external magnetic field, for magnetic resonance imaging and for their medical use in hyperthermia treatment. Our goal is to develop a new chemotherapy system, combining an antineoplastic agent and magnetic nanoparticles. The main advantage is that chemical therapy can be combined with hyperthermia effect using magnetic properties of nanoparticles in order to damage cancerous cells. Moreover, magnetic properties of those nanoparticles can be useful for transport to the biological targets and for magnetic resonance imaging during or after the treatment. Thus, multifunctional magnetic nanoparticles have a great potential in medical field and in particular for cancer therapy research. The major aim of our group is to synthesize biocompatible magnetic nanoparticles with a long blood circulation time and covalently attached antineoplastic agents. This paper deals with the synthesis of silica core–shell maghemite nanoparticles, twice functionalized with amino

 Corresponding author. Tel.: +33 1 44 27 3174; fax: +33 1 44 27 32 28.

E-mail address: [email protected] (J.-M. Siaugue). 0304-8853/$ - see front matter & 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2009.02.051

groups and polyethyleneglycol (PEG) chains. Although iron oxide particles are considered as nontoxic, the biological compatibility can be improved by a silica coating [10,11]. In addition, the biomolecules can be covalently attached to the silica shell using the surface amino groups [4]. Indeed, amino–thiol conjugation, Schiff base compound or peptide bonds can be formed via these groups. PEG chains prevent opsonization and generally increase circulation half life in vivo [12,13]. Moreover, this polymer can improve the colloidal stability by steric effect. Abundant literature describes the synthesis of amino [14,15] or PEG [7,16] functionalized silica core–shell magnetic nanoparticles. But, to the best of our knowledge, only one paper mentions the synthesis of amino–PEG-functionalized core–shell nanoparticles using poly(vinyl pyrrolidone) stabilized nanoparticles prepared via a two-step procedure [4]. Our approach is more direct and has been developed with the biocompatibility in mind as a main criterion. Our procedure is a one-pot procedure, which avoids particle aggregation and particle loss during centrifugation steps, as described previously by Yoon et al. [4]. Moreover, we require no preliminary step with a synthetic polymer to ensure stabilization of the nanoparticles during the silica shell synthesis as opposed to Yoon’s procedure. Another critical point is that our procedure starts from citrated maghemite nanoparticles which are well known, well described and widely used in biomedical applications [17]. In this paper, we present the optimization of this one-pot procedure. First, we studied the influence of several factors for the synthesis of non-functionalized core–shell nanoparticles. Then, from optimized parameters, we have developed the synthesis of amino-functionalized and PEG-functionalized core– shell nanoparticles. Lastly, we combine the two functionalities and realize the synthesis of bi-functionalized core–shell nanoparticles.

ARTICLE IN PRESS V. Maurice et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1408–1413

2. Results and discussion 2.1. Non-functionalized core–shell nanoparticles Magnetic nanoparticles were prepared by co-precipitation of Fe2+ and Fe3+ salts under alkaline conditions by the so called Massart’s process [18]. The experimental conditions of the synthesis were chosen in order to obtain a particles size distribution well described by a log normal law of parameters d0 and s. An aqueous dispersion of maghemite nanoparticles stable in acid or basic conditions was obtained with a point of zero charge near pH 7.3 and citrate anions grafted to their surface in order to have surface charges at biological pH [19]. Silica-coated particles also have negative surface charges at pH 7 and allow in addition the grafting of amino groups. Silica core–shell g-Fe2O3 nanoparticles were prepared from citrate-coated maghemite nanoparticles. Silica shell was formed by the condensation of tetraethoxysilane (TEOS) compound in a mixture of ethanol and water [20]. The presence of citrate allows increasing organosilane affinity for particle surface. Preliminary studies have shown that TEOS condensation with non-citrated nanoparticles leads to non-redispersible suspension. Polymerization was induced by addition of a small amount of ammonia as a catalyst. The reaction of silica condensation is carried out over a night and particles suspension was destabilized by diethyl ether. A red precipitate was formed and separated by magnetic decantation. The precipitate was twice washed with a mixture of diethyl ether and ethanol (15:1). Precipitate was dispersed in Tris/HCl buffer pH 7.4, 100 mM. The final product consisted of a colloidal suspension, which stayed stable for months. Four parameters influencing core–shell synthesis have been studied: ammonia concentration, organosilane concentration, nanoparticles concentration and water to ethanol ratio. Core–shell dispersions were characterized by dynamic light scattering (DLS), infrared spectroscopy, zeta potential measurements at various pH and transmission electronic microscopy (TEM).

Table 1 Mean hydrodynamic diameter and polydispersity index of non-functionalized core–shell nanoparticles. TEOS concentration (mol/L)

Mean hydrodynamic diameter (nm)

Polydispersity index

0.0045 0.009 0.018 0.036 0.072 0.14 0.22

94 97 107 125 128 233 261

0.23 0.23 0.20 0.16 0.11 0.16 0.16

1409

From a given nanoparticles concentration (0.14 g of gFe2O3/L), ammonia concentration clearly controls the quality of the coating. Indeed, a low concentration of ammonia led to the formation of a non-uniform shell around the particles. A minimal amount of ammonia was then necessary to obtain a complete coating of particles and a good dispersion of core–shell particles. Nevertheless, a larger amount of ammonia led to the formation of maghemite free silica particles as a result of rapid hydrolysis of TEOS [20]. An ammonia concentration of 0.12 mol/L appeared to be a good compromise. The second parameter was TEOS concentration (Table 1). For an optimized ammonia concentration of 0.12 mol/L and a given nanoparticles concentration (0.14 g of gFe2O3/L), the minimal concentration of TEOS necessary to ensure a complete covering of particles corresponded to 4.5  103 mol/L. When TEOS concentration was increased, DLS measurements showed a decrease of the polydispersity and of the proportion of small particles. Nevertheless, beyond a given TEOS concentration (0.072 mol/L), the polydispersity of the suspension stayed constant and the mean hydrodynamic diameter increased. Core–shell nanoparticles formed with higher TEOS concentration were not stable under magnetic field anymore neither under gravity. Thus, core–shell synthesis is extremely dependant on the organosilane concentration. We chose a concentration of TEOS equal to 0.072 mol/L, leading to the synthesis of core–shell nanoparticles with a mean hydrodynamic diameter of 128 nm and with a narrow size distribution. The two last factors are dependant on each other. An increase of the nanoparticles concentration implies an increase of the water proportion. Indeed, water stabilizes particles in solution hence an increase of the particle concentration induces an increase of the water amount. With a too small amount of water, maghemite nanoparticles form agglomerates and the synthesis led to the formation of silica core–shell nanoparticles with a high mean hydrodynamic diameter, which was not colloidally stable. Nevertheless, too much water decreased the quality of coating. Indeed, TEOS hetero-condensation is preferentially performed in a mixture of ethanol and water, the ratio between the volume of ethanol and the volume of water depending on the nanoparticles concentration. It was set to 4 for a nanoparticles concentration equal to 0.14 g of gFe2O3/L. When using higher nanoparticles concentrations, 0.27 or 0.48 g of gFe2O3/L, it was necessary to work with a volume ratio of ethanol to water equal to 3 and 2, respectively. The DLS results obtained for the core–shell nanoparticles synthesized with these three nanoparticle concentrations using the optimal conditions for ammonia and TEOS are reported in Table 2. When the nanoparticle concentration increased, the mean hydrodynamic diameter increased (138 nm for 0.27 g of gFe2O3/L and 142 nm for 0.48 g of gFe2O3/L) while the size distribution became slightly larger. Transmission electronic microscopy showed that core–shell nanoparticles have a mean physical diameter of 40 nm and

Table 2 Mean hydrodynamic diameter of non-functionalized, amine-functionalized, PEG-functionalized and PEG/amine-functionalized core–shell nanoparticles. Nanoparticles concentration (g of gFe2O3/L)

Mean hydrodynamic diameter (nm) Nonfunctionalized

0.14 0.27 0.48

128 138 142

Amine-functionalized

PEG-functionalized

PEG/amine-functionalized

10%

20%

10%

20%

10%

20%

244 122 285

117 118 545

80 73 59

66 63 52

86 89 70

75 73 64

ARTICLE IN PRESS 1410

V. Maurice et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1408–1413

lized core–shell nanoparticles were isolated and dispersed in Tris/HCl buffer pH 7.4, 100 mM, as described for the nonfunctionalized core–shell nanoparticles. Zeta potential measurements confirmed the presence of amino groups after this last step: from pH 3 to 9, the zeta potential was positive (Fig. 2) which is in good accordance with the amine pKa data (pKa ¼ 10–11). Infrared spectroscopy confirms the presence of amino groups. The spectrum (Fig. 3, spectrum B) showed shifts of wide bands located around 3000 and 1600 cm1 which are due to the characteristic bands of N–H vibrations. DLS measurements showed in some conditions a decrease of the particles hydrodynamic diameter down to 20 nm (Table 2). For the lower nanoparticle concentration, particles synthesized with 20% of APTS were smaller than those obtained with 10% APTS. Nevertheless, for the higher nanoparticle concentration, DLS measurements indicated the opposite characteristic: particles obtained with 10% APTS have a lower hydrodynamic diameter than those obtained with 20% APTS. For a small concentration of particles, amino groups stabilize particles by electrostatic repulsion and induce a decrease of the particles hydrodynamic diameter. However for a strong proportion of particles, high APTS concentration leads to the formation during the synthesis of silica core–shell nanoparticles with a high mean hydrodynamic diameter.

contain approximately two to five maghemite nanoparticles (Fig. 1). Zeta potential measurements clearly showed that particles are negatively charged (Fig. 2) and infrared spectroscopy confirms the presence of a silica shell (Fig. 3, spectrum A). The region between 200 and 600 cm1 is associated with bending vibration of Si–O–Si bonding while the region after 900 cm1 corresponds to stretching vibration Si–O. The bands around 500, 800, 1100, 1650 and 1900 cm1 are characteristic of silica compounds.

2.2. Amino-functionalized core–shell nanoparticles The first functionality (amine function) was introduced onto the particles obtained as described above by condensation of 3aminopropyltriethoxysilane (APTS) [15]. The organosilane amounts were the same as in the protocol used for the synthesis of non-functionalized core–shell nanoparticles but 10% or 20% of TEOS were substituted with APTS. First, the condensation of TEOS was realized as described above. Then, an equimolar amount of TEOS and APTS was added to the solution which was mixed overnight. The amino-functiona-

2.3. PEG-functionalized core–shell nanoparticles PEG-functionalized core–shell nanoparticles can be synthesized as APTS-modified particles, replacing 10% or 20% of TEOS by PEG instead of APTS. PEG-functionalized nanoparticles are in this case stabilized against aggregation by steric effect, and indeed the mean hydrodynamic diameters were strongly smaller (around 60 nm) than the one of non-functionalized silica core–shell nanoparticles (Table 2) or the one of NH2functionalized particles. DLS results indicate a decrease of particles size when particles concentration increases (Table 2). Zeta potential measurements show that particles stay negatively charged, but with a zeta potential that is less sensitive to pH modification. Thus, electrostatic repulsions also took part in the interaction balance (Fig. 2). Infrared spectroscopy showed the presence of the CH2 stretching vibration bands around 2900 and 3000 cm1 exclusively due to PEG chains (Fig. 3, spectrum C).

Fig. 1. TEM picture of non-functionalized core–shell SiO2–Fe2O3 nanoparticles.

50

Zeta potential (mV)

40 30 20 10 0 -10 -20 -30 -40

3

4

5

6

7

8

9

10

pH Fig. 2. Zeta potential measurements at different pH for non-functionalized (full line), amine-functionalized (mixed line), PEG-functionalized (broken line) and PEG/aminefunctionalized (dotted line) core–shell nanoparticles.

ARTICLE IN PRESS V. Maurice et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1408–1413

2.4. Bi-functionalized core–shell nanoparticles To combine stealthy properties to chemical conjugation possibilities, bi-functionalized core–shell nanoparticles were prepared by the same procedure as above but replacing 10% or 20% of TEOS organosilane by a 50–50 mixture of APTS/PEOS. Bi-functionalized particles have similar properties than PEGfunctionalized nanoparticles: an important decrease of the hydrodynamic diameter compared to non-functionalized particles (Table 2) and a decrease as important as the particles concentration increases. However it has to be noted that, compared to the PEG-functionalized core–shell nanoparticles, the hydrodynamic diameter was slightly higher. Zeta potential measurements

1411

showed that bi-functionalized particles were positively charged from pH 3 to 9 with a point of zero charge located after pH 10. Infrared spectroscopy (Fig. 3, spectrum D) showed the presence of silica bending and stretching vibrations and bands around 2900 and 3000 cm1 characteristic to PEG chains were also identified. Moreover, compared to spectrum B, band shift around 1600 cm1, attributed to N–H vibration, was also present in this spectrum. Finally, magnetic measurements applied to bi-functionalized core–shell nanoparticles were performed using a classical Foner device. The variation of magnetization with applied field for core–shell nanoparticles and citrated nanoparticles is presented in Fig. 4. The value of Ms saturation magnetization obtained for core–shell nanoparticles was similar to citrated nanoparticles,

1.0

Absorbance Units

0.8

0.6

0.4

0.2

3950 3800 3650 3500 3350 3200 3050 2900 2750 2600 2450 2300 2150 2000 1850 1700 1550 1400 1250 1100 950

800

650

500

350

550

400

Wavenumber cm-1

0.40 0.35

Absorbance Units

0.30 0.25 0. 0.15 0.10 0.05

3700 3550 3400 3250 3100 2950 2800 2650 2500 2350 2200 2050 1900 1750 1600 1450 1300 1150 1000

850

700

Wavenumber cm-1 Fig. 3. Infrared spectra of (A) non-functionalized, (B) amine-functionalized, (C) PEG-functionalized and (D) PEG/amine-functionalized core–shell nanoparticles.

ARTICLE IN PRESS 1412

V. Maurice et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1408–1413

Absorbance Units

0.3

0.2

0.1

0.0

3900 3750 3600 3450 3300 3150 3000 2850 2700 2550 2400 2250 2100 1950 1800 1650 1500 1350 1200 1050 900 Wavenumber cm-1

750

600

450

3900 3750 3600 3450 3300 3150 3000 2850 2700 2550 2400 2250 2100 1950 1800 1650 1500 1350 1200 1050 900 Wavenumber cm-1

750

600

450

0.4

Absorbance Units

0.3

0.2

0.1

0.0

Fig. 3. (Continued)

approximately equal to 3  105 A/m, as expected considering silica shell thickness.

3. Conclusion Our work reports the one-pot synthesis of bi-functionalized magnetic core–shell nanoparticles from citrated maghemite nanoparticles. We have synthesized different kind of functionalized silica core–shell maghemite nanoparticles and studied the influence of several parameters in order to optimize synthesis. The

amino functions lead to positive surface charges as shown by zeta potential measurements. The PEG functions increase the steric repulsions and lead in all the cases to a strong decrease of the hydrodynamic diameter down to 70 nm, which is a very good range of size for magnetically assisted separation procedures. These bi-functionalized nanoparticles can be used in applications where colloidal stability is critical. More precisely, these multifunctional magnetic nanoparticles can be grafted with antineoplastic molecules, and thus used as a new chemotherapy agent. Our future works will be dedicated to this topic.

ARTICLE IN PRESS V. Maurice et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1408–1413

1413

Normalized magnetization M/Ms

1.2

1

0.8

0.6

0.4

0.2

0 0

100

200

300

400

500

600

700

800

Magnetic field H (kA.m-1) Fig. 4. Magnetization curves at room temperature for: citrated g-Fe2O3 nanoparticles (full line) and PEG/amine-functionalized core–shell nanoparticles (broken line).

Acknowledgement The authors would like to thank Delphine Talbot and Aude Michel for technical assistance. References [1] S. Mornet, S. Vasseur, F. Grasset, et al., Prog. Solid State Chem. 34 (2006) 237. [2] P. Tartaj, M.P. Morales, T. Gonzalez-Carreno, et al., J. Magn. Magn. Mater. 290–291 (2005) 28. [3] V. Salgueirino-Maceira, M. Correa-Duarte, Adv. Mater. 19 (2007) 4131. [4] T.-J. Yoon, K.N. Yu, E. Kim, et al., Small 2 (2006) 209. [5] N. Sounderya, Y. Zhang, Recent Patents Biomed. Eng. 1 (2008) 34. [6] C.-W. Lu, Y. Hung, J.-K. Hsiao, et al., Nano Lett. 7 (2007) 149. [7] T.-J. Yoon, J.S. Kim, B.G. Kim, et al., Angew. Chem. Int. Ed. 44 (2005) 1068.

[8] J.-P. Fortin, C. Wilhelm, J. Servais, et al., J. Am. Chem. Soc. 129 (2007) 2628. [9] J. Dobson, Drug Dev. Res. 67 (2006) 55. [10] M. Ma, Y. Zhang, W. Yu, et al., Colloid Surf. A—Physicochem. Eng. Asp. 212 (2003) 219. [11] D.K. Yi, S.T. Selvan, S.S. Lee, et al., J. Am. Chem. Soc. 127 (2005) 4990. [12] M.J. Roberts, M.D. Bentley, J.M. Harris, Adv. Drug Deliv. Rev. 54 (2002) 459. [13] D.E. Owens III, N.A. Peppas, Int. J. Pharm. 207 (2006) 93. [14] X. Gao, K.M. Kerry Yu, K.Y. Tam, S.C. Tsang, Chem. Commun. (2003) 2998. [15] S. Mohapatra, N. Pramanik, S. Mukherjee, et al., J. Mater. Sci. 42 (2007) 7566. [16] Y. Zhang, N. Kohler, M. Zhang, Biomaterials 23 (2002) 1553. [17] M. Racuciu, D.E. Creanga, A. Airinei, Eur. Phys. J. E 21 (2006) 117. [18] J.-C. Bacri, R. Perzynski, D. Salin, et al., J. Magn. Magn. Mater. 62 (1986) 36. [19] N. Fauconnier, J.N. Pons, J. Roger, A. Bee, J. Colloid Interface Sci. 194 (1997) 427. [20] Y.-H. Deng, C.-C. Wang, J.-H. Hu, et al., Colloid Surf. A—Physicochem. Eng. Asp. 262 (2005) 87.