Synthesis and characterization of gadolinium nanostructured materials ...

J Nanopart Res (2010) 12:1285–1297 DOI 10.1007/s11051-010-9848-y

RESEARCH PAPER

Synthesis and characterization of gadolinium nanostructured materials with potential applications in magnetic resonance imaging, neutron-capture therapy and targeted drug delivery Dimitrios Stefanakis • Demetrios F. Ghanotakis

Received: 20 November 2009 / Accepted: 2 January 2010 / Published online: 19 January 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Two Gadolinium nanostructured materials, Gd2(OH)5NO3 nanoparticles and Gd(OH)3 nanorods, were synthesized and extensively characterized by various techniques. In addition to the potential use of Gd2(OH)5NO3 in magnetic resonance imaging (MRI) and Neutron-capture therapy (NCT) application, it could also be used in targeted drug delivery. An antibiotic (nalidixic acid), two amino acids (aspartic and glutamic acid), a fatty acid and a surfactant (SDS) were intercalated in the nanoparticles. The surface of the nanoparticles was modified with folic acid in order to be capable of targeted delivery to folate receptor expressing sites, such as tumor human cells. Keywords Gadolinium hydroxides
Folate
Paramagnetic
Antibiotic
Intercalation
Drug delivery
Nanomedicine
Tumor treatment

Introduction Over the last decades contrast-enhanced magnetic resonance imaging (MRI) has become a powerful technique for medical diagnosis and for providing valuable information. MRI contrast agents that

D. Stefanakis
D. F. Ghanotakis (&) Department of Chemistry, University of Crete, 71003 Voutes, Heraklion, Greece e-mail: [email protected]

contain rare-earth elements are widely used in biomedical research and diagnosis. In addition, there is a large number of inorganic nanoparticles that can be potentially used as carriers for the cellular delivery of various molecules such as drugs, genes, and proteins. The combination of the above materials with the rare-earth elements will open new prospects for selective treatment of local tissues and in time diagnosis (Xu et al. 2006). Neutron-capture therapy (NCT) is a cancer therapy, which has gained intense interest in recent years (Sloway et al. 1998; Barth et al. 1999; Coderre and Morris 1999). This therapy uses radiation emitted from gadolinium-157 (157Gd) as a result of its neutron-capture reaction with thermal neutrons. The 157 Gd has some advantages over Boron-10, typically used as radiation producing element in NCT (Barth and Soloway 1994; Mishima et al. 1989); 157Gd has the highest thermal neutron-capture cross section and has been widely used as a magnetic resonance imaging (MRI) contrast agent. Therefore, it could be possible in the future to integrate MRI and NCT (Khokhlov et al. 1996). However, the poor selective tissue labeling and localization provided by conventional molecular Gd chelates has limited success in NCT applications. Methods for encapsulating Gd into nanoparticulate materials have been developed to overcome these limitations (Sharma et al. 2007). Very promising nanoparticles for these applications are the layered double hydroxides (LDH), which are a class of lamellar compounds having the

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general formula MII1-xMIII (OH)2(An-)x/n
mH2O, x II where M is a bivalent and MIII a trivalent anion. These materials are structurally similar to the mineral brucite, Mg(OH)2 with a fraction of M(II) ions replaced by M(III) ions. This replacement results in a positively charged net on the octahedral layers, which is balanced by exchangeable interlayer anions. McIntyre et al. (2008) presented a family of anion exchange intercalation hosts with the generalized formula Ln2(OH)5NO3
xH2O (Ln = Y, Gd–Lu), which contained smaller lanthanide cations as well as approximately hexagonal plate like morphology. The exchange was demonstrated by using a range of organic carboxylate and sulfonate anions (McIntyre et al. 2008). New lanthanide hydroxyhalides Ln2(OH)5X
1.5H2O (X = Cl, Br and Ln = Y, Dy, Er, Yb), have been synthesized hydrothermally. The majority of the materials were obtained as pure phases; however, the Yb materials were always biphasic. Single-crystal diffraction studies showed that one Yb polymorph is monoclinic while the other is orthorhombic. These structures are the first determined for the m = 1 members of the Ln2(OH)6-m(A)m
nH2O family of intercalation hosts. Yb2(OH)5Br
1.5H2O is biphasic displaying larger interlayer separations of 8.33 and ˚ although in this case Y2(OH)5Br
1.5H2O 8.77 A forms the phase with the smaller interlayer spacing (Poudret et al. 2008). A nine layered rare-earth hydroxide with a composition of Ln8(OH)20Cl4
nH2O (Ln = Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, and Y) was recently demonstrated. These phases can be formed through homogeneous precipitation of LnCl3
xH2O with hexamethylenetetramine. All phases are of high crystallinity and are well developed, but they are very sensitive to humidity conditions (Geng et al. 2008). Gd(OH)3 is consisted of bundle-like nanorods and has numerous applications in science and technology, due to its special optical, electrical, and chemical properties (Rao et al. 1997). The structure of Gd(OH)3 is hexagonal P63/m and the gadolinium atoms array on parallel linear chains. Each chain is surrounded by three other chains and form hexagonal tunnels. In the present study, we report the synthesis of two type materials, Ln2(OH)5NO3 (Ln = Gd and Dy) and Gd(OH)3. Ln2(OH)5NO3 belongs to a new class of anion exchangeable layered hydroxides with similar characteristics to LDH. Hydroxynitrate phases,

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Ln2(OH)6-m(NO3)m
nH2O, are known for the lanthanide cations. Organic molecules were intercalated in Ln2(OH)5NO3 in order to make it an attractive drug carrier. In particular, an antibiotic (nalidixic acid), two amino acids (aspartic and glutamic acid), a fatty acid and a surfactant (SDS) were intercalated. The as-synthesized materials were investigated by employing various techniques, such as powder X-ray diffraction, FT-IR spectroscopy, thermogravimetric analysis, TEM, SEM, and HRTEM microscopy, ED, and magnetic measurements.

Materials and methods Synthesis of Ln2(OH)5NO3
xH2O The Ln2(OH)5NO3 (Ln = Gd and Dy) precipitate was prepared by the standard aqueous coprecipitation method. 30 mL of a solution containing 0.225 M Ln(NO3)3 and 0.675 M Mg(NO3)2 and 15 mL of a solution containing 0.5 M NaOH were added dropwise to 50 mL of H2O with vigorous stirring under nitrogen gas flow. The flow rate of the two solutions was adjusted so that the pH was equal to 7.0 ± 0.5 throughout the addition. All solutions were prepared using deionized water and were purged of CO2 using nitrogen gas for 20 min. After the end of the addition, the suspension was treated hydrothermally at 120 °C for 24 h under reflux conditions. The resulting white suspension was centrifuged at 15,0009g for 15 min, washed twice with deionized and CO2-free water and finally was dried in air at 100 °C. Modification of Gd2(OH)5
xH2O nanoparticles with folic acid 100 mg dried nanoparticles were dispersed in 50 mL toluene containing 3 mM (3-aminopropyl)-trimethoxysilane. The mixture was vortexed, sonicated, and incubated at 60 °C for 4 h. The suspension was centrifuged and washed with methanol. The dried precipitates were added to 40 mL dimethylsulfoxide (DMSO), which contains 10 mM folic acid, 0.25 mM DCC, and 0.025 mM pyridin. The reaction carried out at 37 °C for 4 h. Afterward, the suspension was centrifuged and the precipitate was washed with DMSO.

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Synthesis of Gd(OH)3 The Gd(OH)3 was synthesized similarly by a hydrothermal process. A mixed solution of 0.225 M Gd(NO3)3 and 0.225 M Mg(NO3)2 was added dropwise to 50 mL H2O under stirring and nitrogen atmosphere. Solution pH was adjusted to 10 by dropwise addition of 1 M NaOH solution. After ending of the addition the suspension was heated at 120 °C for 24 h under reflux conditions. The resulting white suspension was centrifuged at 15,0009g for 15 min, washed twice with deionized and CO2-free water and finally was dried in air at 100 °C. Organic anion exchange derivatives of Gd2(OH)5NO3 The organic anions were intercalated by simply mixing the aqueous solutions of them with the Gd2(OH)5NO3. The solutions were prepared using deionized, CO2-free water. After the reaction the nanohybrid products were collected by centrifugation at 12,0009g for 15 min, washed twice with water, filtered under vacuum and dried in air flow. Gd2(OH)5NO3-aspartic To prepare Gd2(OH)5NO3-aspartic acid, 0.30 g (45 mmol) of the amino acid was added to water containing 50 mM Tris as a buffer and the pH of the solution was then adjusted to 10.0. Then, 0.1 g of Gd2(OH)5NO3 suspension was added and the solution (final volume 50 mL) was stirred at room temperature for 24 h. Gd2(OH)5NO3-glutamate 100 mg (13.6 mmol) of Glutamic acid was added in an aqueous solution of sodium Tris (10 mmol) pH 9.5. Finally, the appropriate volume of the Gd2(OH)5NO3 suspension equal to 0.10 g of Gd2(OH)5NO3was added (final reaction volume 50 mL) and the solution was stirred at room temperature overnight.

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X-100 (0.04 g, 1% w/w) buffered at pH 9.0 with 10 mM Tris. Finally, the appropriate volume of the Gd2(OH)5NO3 suspension equal to 0.20 g of Gd2(OH)5NO3 was added (final reaction volume 400 mL) and the solution was stirred at room temperature overnight. Gd2(OH)5NO3-SDS To obtain this nanohybrid, 1.44 g (0.1 mmol) of SDS was dissolved in water containing 10 mM Tris as a buffer. The pH of the solution was then adjusted to 7.0 and an appropriate amount of Gd2(OH)5NO3 suspension containing 0.20 g of it was added. The solution (final volume 50 mL) was stirred at room temperature for 24 h. Synthesis of Gd2(OH)5-nalidixic acid nanohybrid Gd2(OH)5-nalidixic acid nanohybrid was synthesized according to the above precipitation method. Two aqueous solutions, one containing a mixture of Gd(NO3)3 and Mg(NO3)2 and the other containing NaOH, were added dropwise to a solution of nalidixic acid, while the pH was maintained at 7. The resulting slurry was heated at 60 °C for 24 h. The precipitate was washed with distilled water and then dried. Orientation of Gd particles onto Si wafer For the attachment of Gd2(OH)5NO3 particles, we employ clean silicon (100) wafers as a substrate. Gd2(OH)5NO3 and folate modified Gd2(OH)5NO3 particles were suspended in dry organic solvent, such 1-propanol, in a covered laboratory glass flask and then immersed in an ultrasonic bath for 6 h at room temperature. The Si wafers were dipped in the colloidal solution and sonicated for another 2 h. Previously, Si wafers were treated with a solvent mixture H2SO4:H2O2 (4:1) to obtain a Si surface passivating oxide (Kayambaki et al. 1995). The Gd2(OH)5NO3-coated Si wafer was removed and underwent sonication for an additional hour to remove the excess Gd2(OH)5NO3 particles from the substrates.

Gd2(OH)5NO3-palmitic acid

Characterization

Palmitic acid (0.36 g, as a 36 mg/mL solution in ethanol) was added in an aqueous solution of Triton

Powder X-ray diffraction (XRD) patterns were recorded on a Rigaku RINT 2000 powder

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˚´ ) diffractometer, using Cu Ka radiation (k = 1.54 A at 40 kV and 178 mA. FTIR spectra were recorded on a Thermo-Electron Nicolet 6700 FTIR optical spectrometer with a DTGS KBr detector. Differential thermal analyses (DTA) and thermogravimetric analyses (TGA) were carried out by using the Perkin-Elmer DTA7 and TGA7 instruments, respectively. The analyses were performed in nitrogen atmosphere at a heating rate of 10 °C/min. Simultaneous TG–MS analysis was performed in a Pyris Diamond TG–DTA coupled to ThermoStarTM QM220 mass spectrometer by a quartz capillary transfer line. Transmission Electron Microscopy (TEM) Micrographs were recorded with a JEOL JEM 100C— electron microscope operating at 80 kV, equipped with a Model 782 ES500W Erlangshen CCD camera. A multimode AFM (Digital Instruments Nanoscope IIIa) was used to study the AFM phase images. In order to examine the average stoichiometry of the particles, we used an energy-dispersive X-ray analysis (EDX) system attached to a scanning electron microscope (Philips SEM 535 with EDAX equipment).

Results and discussion Gd(III) is a good paramagnetic metal ion because of its seven unpaired electrons, symmetric electronic states, high relaxivity and a total coordination number of nine. However, due to their undesirable

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biodistribution and relatively high toxicity, paramagnetic metals cannot be used as contrast agents in their ionic form. Thus, we tried to synthesize paramagnetic nanoparticles, based on LDH, that contain lanthanide atoms. Due to size restrictions, the number of cations that can be incorporated into LDH is relatively limited. Consequently, there have not been any reports of LDH containing gadolinium cations or other lanthanides. When we tried to synthesize hydrotalcite like compounds, with Gd(III) and Ca(II) cations in the layers, according to the Miyata (1975) protocol at pH = 10, we observed the existence of two phases. The XRD patterns revealed that these phases consist of Gd(OH)3 and a lamellar compound with a series of strong (00l) peaks (Fig. 1c), similar to LDH. In order to separate these phases, we repeated the synthesis under a series of different conditions. We varied the cations and the pH and, as shown in Table 1, we managed to find the conditions that lead to the synthesis of each phase in its pure form (Fig. 1a, b). The ratio of Gd(III) to the second cation was also investigated and it was found that the best results were obtained with a ratio of 1:2 to 1:3. X-ray and EDS studies showed that the formula of the second phase is Gd2(OH)5NO3. This product is characterized by high stability and solubility for many weeks. EDS studies demonstrated the absence of magnesium atoms from the sample of Gd2(OH)5NO3 (Fig. 2a). These results confirm that Gd2(OH)5NO3 is similar to LDH, however, it cannot be classified in the same family, because LDH consists of two

Fig. 1 Powder X-ray diffraction pattern of (a) Gd(OH)3, (b) Gd2(OH)5NO3, (c) both phases, A and B, and (d) Dy2(OH)5NO3

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J Nanopart Res (2010) 12:1285–1297 Table 1 Experiment conditions of synthesis Gd(OH)3 & Gd2(OH)5NO3 Cations Gd3? & Ca2? Gd

3?

Result

10

Gd(OH)3 & Gd2(OH)5NO3

10

Gd(OH)3 & Gd2(OH)5NO3

Gd & Gd3?

10

Gd(OH)3 & Gd2(OH)5NO3

Gd3? & Mg2?

10

Gd(OH)3

Gd3? & Mg2?

7

Gd2(OH)5NO3

Gd3? & Na?

7

Gd2(OH)5NO3

7

Gd2(OH)5NO3

7

Neither of them

Gd

3?

Gd3?

& Na

?

pH

& Ca

2?

cations, a bivalent and a trivalent, while Gd2(OH)5 NO3 contains only gadolinium(III). It is interesting to note that when Gd(III) and Mg(II) are used for the synthesis, depending on the pH we obtain either Gd(OH)3 (pH 10) or Gd2(OH)5NO3 (pH 7). Actually, this is the final protocol we used for the synthesis of the nanostructured materials that have been used throughout this study.

Characterization of Gd2(OH)5NO3 and Gd(OH)3 The XRD pattern of Gd2(OH)5NO3 exhibits the characteristic reflections of LDH materials with a series of strong (00l) peaks appearing as narrow symmetric lines at low angle as well as higher order reflections. These peaks correspond to the basal ˚ (Fig. 1b). Furthermore, a spacing, which is 9.11 A significant number of weak non-basal reflections are observed, due to the fact that the layers are ordered. In contrast to Gd2(OH)5NO3, these reflections could not be observed for LDHs. According to Scherrer’s formula the thickness of the crystallite was estimated ˚. to be 394 A Dy2(OH)5NO3 was also synthesized, by using the protocol developed for the preparation of Gd2(OH)5 NO3. The X-ray diffraction pattern revealed that Dy2(OH)5NO3 is similar to Gd2(OH)5NO3, appearing ´˚ highly organized peaks with basal spacing 9.13 A (Fig. 1d). Figure 1a shows the powder X-ray diffraction pattern of the as-prepared Gd(OH)3 product. The XRD peaks correspond to the hexagonal structure of ˚ and Gd(OH)3 with lattice constant a = 6.3 A ˚ . The peaks which were characterized with c = 3.6 A high intensity in addition to the narrow peaks,

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demonstrate a high crystal organization. According to Scherrer’s formula the thickness of the crystallite ˚ . The ED spectra confirm the presence of is 417.5 A only gadolinium and oxygen atoms (Fig. 2b) in the Gd(OH)3 material. The FT-IR spectrum of Gd2(OH)5NO3 is shown in Fig. 3b. Characteristic of this spectrum is the strong and broad absorption band centered around 3,590 cm-1 which is attributed to the superposition of O–H stretching bands v(OHstr) arising from metalhydroxyl groups, and hydrogen-bonded interlayer water molecules. Another absorption band due to the water deformation, d(H2O), is recorded around 1,630 cm-1. The presence of the NO3- anion is revealed by the sharp characteristic peak of NO3observed at 1,350 cm-1. The band observed in the low-frequency region of the spectrum, corresponds to the lattice vibration mode and may be attributed to Gadolinium oxygen (Gd–O) vibration at 520 cm-1. The FT-IR spectrum of Gd(OH)3 includes two sharp bands at 3,610 and 3,698 cm-1, which are associated to the symmetrical and asymmetrical stretching vibration of O–H group (Fig. 3a). The band corresponding to the vibration mode dHOH appears at 1,634 cm-1. The bands below 1,000 cm-1 are Gd–O vibration modes. The thermogravimetric analysis (TGA) curve of Gd2(OH)5NO3 is comparable to the one observed for the layered hydroxide materials (McIntyre et al. 2008). A typical TGA curve is shown in Fig. 4a. The results show an initial reduction in weight (7.69%) between room temperature and 150 °C, which arises from the interlayer water. A second weight loss between 150 and 320 °C results from a concomitant dehydroxylation of the inorganic layers, the reduction of nitrates to nitrites and the decomposition of the layers (Xu et al. 2001; Lo´pez-Salinas et al. 1996). Beyond 320 °C, a further condensation of hydroxyls and a complete decomposition is observed. The thermal behavior of Gd2(OH)5NO3 is characterized by an endothermic transition at 275 °C, that corresponds to the dehydroxylation. The TGA of Gd(OH)3 exhibits three stages of weight loss (Fig. 4b). The first weight loss occurs below 100 °C and has been attributed to the removal of interlayer water. The second weight loss recorded in the temperature range of 270–310 °C was ascribed to the initial dehydroxylation of the inorganic nanorods. The final weight loss occurs in the temperature

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Fig. 2 ED spectra of a Gd2(OH)5NO3 and b Gd(OH)3

Fig. 3 FITR spectra of (a) Gd(OH)3, (b) Gd2(OH)5NO3, (c) Gd2(OH)5NO3–NH2 and (d) Gd2(OH)5NO3–NH2-folate

range of 320–430 °C and corresponds to the final dehydroxylation of the sample. In the case of Gd(OH)3 three endothermic transitions were observed. The first transition occurs at 50 °C, the second one at 300 °C and the third one at 375 °C.

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TEM micrographs of Gd2(OH)5NO3 particles are shown in Fig. 5a. These images exhibited small accumulations of individual plates, oriented parallel to the grid surface. These platelets have an average thickness of 50 ± 5 nm. High resolution transmission electron microscopy (HRTEM) images confirm the high crystallinity of Gd2(OH)5NO3; these images show a lamellar structure with a repeating fringe of 0.31 nm (Fig. 5b, c). The view of their edge confirms that they are single hexagonal platelets, resembling the morphology of typical LDHs (Kriven et al. 2004). The general SEM morphologies of Gd(OH)3 are shown in Fig. 6b. It can be seen that the product mainly consists of solid rod-like structures. The length of the rods can be up to 400 nm. Figure 6a shows a low-magnification TEM image of Gd(OH)3 nanorods, clearly showing that the product is entirely composed of crystals with a relatively uniform, rodlike morphology. High resolution TEM image shows

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Fig. 4 TGA analysis of a Gd2(OH)5NO3 and b Gd(OH)3

Fig. 5 a TEM images of Gd2(OH)5NO3, b and c HRTEM images of Gd2(OH)5NO3

that the nanorods are nearly structurally uniform, and the clear lattice fringes illustrate that the nanorod is a single crystalline. The interplanar spacing (Fig. 6c) is about 0.294 nm, which corresponds to [1 0 1] planes. Since Gadolinium’s prominent feature is the high number of unpaired electrons, Gd2(OH)5NO3 is expected to have paramagnetic properties. In accordance with the crystal structure, Gd2(OH)5NO3 exhibits a simple Curie paramagnetic behavior with an effective magnetic moment of 7.88 lB, which is similar to the theoretical estimated value (7.94 lB)

(Want et al. 2008). Furthermore, the paramagnetic constants were estimated by the Curie–Weiss law, where v = C/(T - h). In particular, the Curie temperature h and the Curie constant C are -7.774 K and 7.778 emu K/mol, respectively. The magnetic properties of the Gd2(OH)5NO3 composite were studied and the magnetization curve is shown in Fig. 7. As evidenced from the figure, the nanoparticles didn’t show hysteresis at low temperature (10 K), which is typical to nanosized magnetic materials demonstrating superparamagnetic behavior

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Fig. 6 a TEM images of the Gd(OH)3, b HRTEM images of the Gd(OH)3 and c SEM images of the Gd(OH)3

Fig. 7 Magnetization curves of Gd2(OH)5NO3 composite

(Vijayakumar et al. 2000; Abu-Much et al. 2006; Harris et al. 2003). The total saturation magnetization of the composite is 12.8 emu/g. The inverse magnetic susceptibility curves of Gd(OH)3, v-1 = H/M, as a function of temperature for fixed H = 1T are shown in Fig. 8. The constant h

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has a value of -7.29 K and the Curie constant C is 7.28 emu K/mol. Gd(OH)3 exhibits a simple Curie paramagnetic behavior with an effective magnetic moment of 7.28lB. Similar to Gd2(OH)5NO3, Gd(OH)3 nanorods did not show hysteresis at 10 K, demonstrating

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Fig. 8 Magnetization curves of Gd(OH)3 composite Fig. 9 AFM images of a Gd2(OH)5NO3 and b folate-modified Gd2(OH)5NO3 particles adsorbed onto silicon wafer

superparamagnetic behavior at this temperature. The total saturation magnetization of the composite is 20.9 emu/g. Gd2(OH)5NO3 particles were bonded on the surface of the Si wafer by electrostatic forces, as the oxide surface attracts the positive layers of the Gd2(OH)? 5 . Figure 9 presents the AFM image for the as-adsorbed Gd2(OH)5NO3 particles on Si. A random attachment of the particles on the surface of the Si wafer, is observed. In addition, the density of the particles on the surface is proportional to the amount of the initial suspension. A relatively broad distribution of the attached particles size is observed. In particular, the average height of the crystallites was about 5 nm and the surface distance 70 nm,

while the larger particles have dimensions of 50 nm height and 100 nm horizontal distance. Surface modification of Gd2(OH)5NO3 The surface of Gd2(OH)5NO3 was modified in order to obtain water solubility and functionalization. In addition, this coating prevents the nanoparticles forming large aggregates. In particular, the modification with folic acid is a very promising strategy to carry out efficient and specific cellular uptake of nanoparticles, as the surface contains a ligand that is efficiently taken up by target cells via receptormediated endocytosis (Lowry et al. 1998). In contrary to the normal cells, the folate receptors are

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Figure 9b shows the AFM image for folate modified Gd2(OH)5NO3 particles absorbed on Si; the average height of the particles was about 10 nm and the surface distance 90 nm. This orientation of Gd2(OH)5NO3 layers may have potential applications in designing nano-sized sensor, catalysis, and nanosized containers for biomolecules). Organic anion exchange derivatives of Gd2(OH)5NO3

Fig. 10 Powder X-ray diffraction pattern of (a) Gd2(OH)5NO3 modified with APTS and (b) Gd2(OH)5NO3–NH2-folate

overexpressed in various human tumors (Antony 1996; Garin-Chesa et al. 1993; Ross et al. 1994; Lu and Low 2002), where they function by capturing folate, in order to feed rapidly dividing tumor cells (Sudimack and Lee 2000; Xu et al. 2001; Maziarz et al. 1999). Initially, introduction of amine groups on the surface of Gd2(OH)5NO3 is carried out via silanization with APTS. The surface modification with amine groups is mainly focused in the second functionalization with folic acid. The powder X-ray diffraction patterns of Gd2(OH)5NO3 and the coated with amino groups and folic acid are shown in Fig. 10. Although the peak intensity somewhat decreased by modifying the surface of the layers, the peak position hardly changed. FT-IR spectroscopy displays the surface modification of Gd2(OH)5NO3 and the covalent binding with folic acid (Fig. 3c, d). After the APTS reaction, new peaks are detected at 1,120 and 1,056 cm-1, due to the Si–O (Mikhaylova et al. 2004; Wypych et al. 2005). In addition, the NH2 deformation occurs at 1,520 cm-1 (Tonle et al. 2003). The aliphatic C–H stretching vibrations are found in the region between 3,000 and 2,700 cm-1 (Colthup et al. 1975; Socrates 1994). Aliphatic CH2 groups give rise to a doublet at 2,918 and 3,003 cm-1, which is assigned to asymmetric and symmetric stretchings, respectively. A strong and medium absorption at 1,019 and 947 cm-1 attributed to C–C–H symmetric and C–C–N asymmetric bending. A new peak appears at 1,608 cm-1 as a result to the covalent binging of folate on the surface, as the amide II bond is formed.

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Two amino acids, a fatty acid and a surfactant were intercalated into the Gd2(OH)5 host successfully. During ion exchange of the nitrate anions, the layers of Gd2(OH)5 expand to host anions and this expansion is reflected to the d-spacing values that are calculated from the mean value of the first, second and third order peaks of the XRD diagrams (Fig. 11). ˚ for Gd2(OH)5-aspartate and These values are 11.74 A ˚ for Gd2(OH)5-glutamate, 49.02 A ˚ for 11.68 A ˚ for Gd2(OH)5-palmitic Gd2(OH)5-SDS and 27.24 A acid. In contrast, the d-spacing of Gd2(OH)5NO3 is ˚ . SDS surfactant forms bimolecular films in 9.11 A comparison to the other intercalated molecules (You et al.). The presence of the aminoacids in the nanohybrids was also demonstrated by infrared spectroscopy. The characteristic absorptions of the carboxyl group at 1,556 and 1,404 cm-1 is evidence for the intercalation of glutamic acid and aspartic acid in the nanohybrids (data not shown) (Nunes and Cavalheiro 2007). The infrared spectra of the Gd2(OH)5-palmitic acid and of Gd2(OH)5-SDS nanohybrids confirm the successful intercalation into Gd2(OH)5 (data not shown). We also synthesized the Gd2(OH)5-nalidixic acid ˚ obtained from nanohybrid. The d-spacing of 21.42 A the XRD diagram of the nanohybrid Gd2(OH)5nalidixic acid shows the formation of a monolayer of nalidixic acid molecules in the interlayer between the inorganic layers (Fig. 11f). The presence of the antibiotic in the nanohybrids was also demonstrated by infrared spectroscopy (data not shown). The nanohybrid shows a strong absorption at 1,620 cm-1 attributed to the stretching vibration mC=C of the aromatic rings (Neugebauer et al. 2005). The stretching vibration of the ketone at C4 appears at 1,569 cm-1, while the symmetric

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Fig. 11 Powder X-ray diffraction pattern of (a) Gd2(OH)5NO3, (b) Gd2(OH)5-glutamic acid, (c) Gd2(OH)5-aspartic acid and (d) Gd2(OH)5palmitic and (e) acid Gd2(OH)5-SDS and (f) Gd2(OH)5-nalidixic acid

stretch of the 1,400 cm-1.

carboxylate

group

appears

at

Conclusions Because of their properties gadolinium nanostructured material are excellent candidates for future medical applications, such as magnetic resonance imaging (MRI), Neutron-capture therapy (NCT) and targeted drug delivery. In this particular study, we have developed a simple experimental protocol for the preparation of a novel class of layered hydroxide nanosheets, with the general composition Ln2(OH)5NO3
H2O (Ln = Gd and Dy). The nanoparticles were thoroughly characterized by various techniques (powder X-ray diffraction, FT-IR spectroscopy, thermogravimetry, TEM, SEM, and HRTEM microscopy, EDS, and magnetic measurements), and were found to be very flexible host lattices undergoing facile anion exchange reactions at room temperature, with a wide variety of organic molecules. More specifically, two amino acids, a fatty acid, a

surfactant, and an antibiotic were intercalated. Thus, Ln2(OH)5NO3 could potentially be used as inorganic drug carrier for many different pharmaceutically active compounds like amino acids and antibiotics. Since targeted drug delivery is one of the main objectives in pharmaceutical research, we proceeded in modifying the surface of the nanoparticles with folic acid in order to be capable of targeted delivery to folate receptor expressing sites, like tumor human cells. This modification was achieved by covalent bonding of folic acid, via silanization with APTS. McIntyre et al. (2008) have reported the preparation of a series of these novel family materials, containing the smaller lanthanide cations. Although, under their experimental conditions, Gd2(OH)5NO3 was also synthesized, it was less crystalline than the others; it was suggested that gadolinium marks the limiting cation size for this phase. As demonstrated throughout this manuscript, our synthetic protocol results in the preparation a more crystalline Gd2(OH)5NO3 material without impurities. Since Gd2(OH)5NO3, especially the functionalized material, might be an interesting nanoparticle for

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MRI applications, we are planning to carry out MRI studies. In addition to Gd2(OH)5NO3, Gd(OH)3 was also synthesized by a similar hydrothermal method. This rod-like structure material displays a highly ordered structure without impurities. The particles have a diameter of 40–60 nm and a length of around 500 nm; magnetic measurements showed a typical paramagnetic behavior. Since Gd2(OH)5NO3, especially the functionalized material, might be an interesting nanoparticle for diagnostic MRI and targeted drug delivery applications, we are planning to further explore these potential by using various cell lines that overexpress the folate receptors. Acknowledgments This work was part of the 03ED375 research project, implemented within the framework of the ‘‘Reinforcement Programme of Human Research Manpower’’ (PENED) and co-financed by National and Community Funds (25% from the Greek Ministry of Development-General Secretariat of Research and Technology and 75% from E.U.-European Social Fund). The authors would like to thank Dr. A. Lappas (IESL Crete) for the magnetic measurements, Mrs A. Tsagkaraki (University of Crete) for the AFM studies, the Polymer & Colloid Science Group as well as the NanoComplex Materials staff of IESL of Crete for XRD measurements. Finally, we would like to thank the staff of the microscope lab of the Biology department (University of Crete).

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