Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.
A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.
Atherosclerosis is a multifactorial chronic inflammatory disease of the arterial wall resulting from a deregulated lipid metabolism and a defective inflammatory response. Due to the prevalence and the economical and social costs of this and related cardiovascular diseases there is a growing interest in addressing the pathology with new tools, of which nanotechnology is one of the most promising.1-3 However there are very few examples of fast production and characterization of probes which is basic for translation to the clinic.4 In this protocol we use a microwave synthesis of iron oxide nanoparticle for further functionalization with a bisphosphonate and in vivo detection of atherosclerosis in ApoE-/- mice in 1 hr.5 Iron oxide nanoparticles (IONP) are a well-known nanomaterial and its use as a contrast agent for Magnetic Resonance Imaging (MRI) has been established for the detection of different diseases in the last years.6-8
Microwave synthesis (MWS), allows synthesizing nanoparticles in extremely short times with high reproducibility and enhanced yields.9,10 In our protocol we obtain IONP with plaque targeting capabilities in three steps. The final one is the attachment of an aminobisphosphonate, Neridronate, which is key in our strategy due to its calcium-binding properties. Due to their natural analogue pyrophosphate (PPi), Neridronate has been used in the treatment of Osteogenesis Imperfecta (OI) and Paget's disease of bone (PDB) for their high affinity towards bone mineral.11-13
The three steps of the protocol are summarized in scheme 1. Steps one and two are carried out using microwave technology. First step provide oleic acid-coated iron oxide nanoparticles (OA-IONP) by a modification of published methods.14 The protocol is an adaptation to microwave synthesis of the traditional thermal decomposition synthesis.15,16 A mixture containing Fe(acac)3, oleic acid, oleylamine and 1,2-dodecanediol is dissolved in benzyl alcohol and subjected at two heating processes. Purification is carried out washing with EtOH and collecting the particles with a Nd-Fe-B magnet to eliminate the excess of surfactants in the supernatant. Then, OA-IONP are stabilized in CHCl3. As expected, due to the very fast heating, anticipated results showed that the nanoparticles synthesized by microwave are smaller in terms of core (3.7 ± 0.8 nm) and hydrodynamic size (7.5 nm) in comparison with traditional thermal decomposition; however, nanoparticles still present an excellent crystallinity.
The second step consists in a direct chemical modification of the double bond, present in the oleic acid, using a strong oxidant like KMnO4, the original methodology developed in our group was modified for MW conditions.17 A first stage forms the complexes between MnO4– and the double bond. Then, a second stage in acidic conditions, produce the cleavage of the oleic acid molecule giving Azelaic acid-IONP. After these two stages of 9 min each, the sample is purified, first washing with NaHSO3 1% to reduce the excess of MnO4– to MnO2 and then with NaOH 1% to neutralize the acid.
After the purification step, Azelaic-IONP are stabilized in 10 mM phosphate buffer pH = 7.2. This buffer is the best environment for the colloidal stability of the particles similarly to what happened in the original, thermal reaction.18 The use of microwave for the direct oxidation of the double bond contained in OA-IONP is a very good example of the advantages of using this technology in the synthesis of nanoparticles. With the classical method the reaction takes 24 hr, the utilization of microwave decrease the reaction time to 18 min. Moreover, the microwave-driven protocol shows an excellent reproducibility giving nanoparticles with 30 ± 5 nm of hydrodynamic size after 4 repetitions. Apart of the change in the hydrodynamic size, the zeta potential is a good parameter to quickly check the successful of the reaction. Due to the presence of the new carboxylic groups in Azelaic-IONP, the value for the zeta potential is around -44 mV, very similar to the value obtained by the thermal approach.
For the attachment of neridronate to Azelaic-IONP, traditional EDC/sulfo-NHS conjugation is used.19 This synthetic approach is well-established since employing an activated carboxylate with the sulfo-NHS ensures colloidal stability during the reaction. After the elimination of phosphate buffer the reaction with neridronate is carried out in 1 mM HEPES buffer (pH ~7). The reaction renders Neridronate-IONP with a hydrodynamic size of 40 ± 4 nm in a narrow size distribution and -24.1 mV of zeta-potential.
The procedure is described for fast synthesis of IONP for in vivo visualization of atherosclerotic plaque although the feasibility of the method allows the attachment of any peptide/antibody with free amines, using the same conditions, for different purposes within T2-weighted contrast agent MRI field.
1. Preparation of Reagents
2. Synthesis of Oleic Acid Coated Nanoparticles (OA-IONP)
3. Synthesis of Azelaic Acid Nanoparticles (Azelaic Acid-IONP)
4. Synthesis of Neridronate Nanoparticles (Neridronate-IONP)
5. In Vivo Detection of Atheroma Plaque in ApoE-/- Mice by MRI
In this protocol, the synthesis of three different IONP is described. Starting from hydrophobic OA-IONP, aqueous stable nanoparticles are obtained with the help of microwave-driven synthesis. All nanoparticles presented ultra-small hydrodynamic size (Dh <50 nm) in a very narrow size distribution (Figure 1c). The use of microwave technology renders ultra-small nanoparticles in terms of core sizes. Since microwave produce a fast heating, the rate of the nucleation increase in comparison with others methodologies giving smaller sizes in the core of the nanoparticles. However, the particles still present excellent crystallinity as is shown in the TEM images where the lattice fringes on the Fe3O4 cores can be clearly seen (Figure 1a, b). Other important aspect of the method is the reproducibility. After four repetitions of the synthesis of Azelaic acid-IONP, the same results in the hydrodynamic size and distribution were obtained (Figure 1d).
After functionalization, the Ca2+ binding properties due to bisphosphonates present in neridronate nanoparticles were checked incubating these nanoparticles with different amounts of Ca2+. It was shown that T2 relaxation time increments linearly with the amount of Ca2+ and the time of incubation due to the formation of clusters of nanoparticle whereas nanoparticles without Ca2+ remained stable (Figure 1e), conforming our initial hypothesis.
In vivo MRI experiments were performed in 48 weeks old ApoE-/- mice. Carotids and abdominal aorta basal images were first taken. Lesion due to the formation of the atherosclerotic plaque can be clearly seen. Then, 100 µl (1 mg [Fe] ml-1) of neridronate nanoparticles were intravenously injected into tail vein and the images acquired 1 hr post injection. As it is shown (Figure 2), 1 hr post injection the signal form the plaque is hypointense in comparison to the basal images. Selection of two ROIs (region of interest) allows the quantification of the intensity signal in the lesion area for the comparison between basal and 1 hr post injection images. The plaque to muscle ratio is significantly different between them (p <0.05, Figure 2b).
In addition, signal in the liver was monitored in mice after injection of 100 µl of neridronate nanoparticles to assess if the reduction of the intensity was due to circulation time in the blood and not by selective accumulation. As the graph shows (Figure 2c), nanoparticles were completely cleared from circulation after 20 min confirming the selective accumulation of neridronate nanoparticles towards atherosclerotic plaque. Final ex vivo imaging and histology were performed. Mice were sacrificed and the aortas extracted. Imaging of aortas with and without nanoparticles showed differences in the signal in agreement with in vivo experiments (Figure 2d).
Scheme 1: Synthetic steps followed in the protocol and basic characterization at each point by DLS. Please click here to view a larger version of this figure.
Figure 1: Characterization of nanoparticles. (a) TEM images, at two magnifications, for OA-IONP; (b) TEM images, at two magnifications, for Neridronate-IONP; (c) hydrodynamic size for nanoparticles OA-IONP, Azelaic acid-IONP and Neridronate-IONP; (d) hydrodynamic size for Azelaic acid-IONP in four different synthesis and (e) evolution of T2 relaxation time in a solution of Neridronate-IONP as a function of time and calcium concentration (ref TEM protocol: NIST – NCL Joint Assay Protocol, PCC-X, Measuring the Size of Nanoparticles Using Transmission Electron Microscopy). Please click here to view a larger version of this figure.
Figure 2: MRI data of the plaque. (a) In vivo MRI of ApoE-/- mouse before (top) and one hour after the i.v. injection of Neridronate-IONP (bottom); (b) plaque to muscle relative signal intensity before (basal) and one hour after the i.v. injection of Neridronate-IONP; (c) liver to muscle relative signal intensity at different time points after the injection of Neridronate-IONP and (d) ex vivo images of the aorta for two mice, with and without the injection of nanoparticles5. Please click here to view a larger version of this figure.
Iron oxide nanoparticles (IONP) are one of the most important nanomaterials and it has been used for different applications from long time ago. The use of these materials as contrast agent for magnetic resonance imaging (MRI) is a well-established field. However, the routes of synthesis often take several time and the setting is complicated. Due to dramatically reduce reaction times and enhances reproducibility the use of microwave-driven synthesis seems to be a good alternative for the production of high quality nanoparticles. In the protocol described above, microwave technology has been used for the synthesis of two different nanoparticles. Microwave allow for a fine-tuning of the main parameters that can affect the final characteristics of the particles. It is important to note that the physical properties of the nanoparticles will change if any of the described conditions are changed. Since some chemicals are used in the synthetic procedure, the purification steps are critical to obtain high quality nanoparticles.
In OA-IONP excess of surfactants are used in order to get enough stability in the nanoparticles. After synthesis, three purification steps are mandatory to remove it. For the synthesis of Azelaic acid-IONP, two different microwave stages are required. In the second stage, the final size of the particles can be tuned from ultra-small IONP (Dh <50 nm) using pH = 2.9 to larger hydrodynamic size (Dh >50 nm) using physiological pH. In the purification of the Azelaic acid-IONP, the amount of NaOH used is essential. Enough amount of NaOH has to be added to stabilize the nanoparticles, however too many NaOH can desorb the surfactant from the nanoparticles rendering unstable material.
Typically, IONP possess short circulation time in blood which is one of the main disadvantages. For its use as contrast agent, nanoparticles need to circulate enough time in blood to reach the desired area. To increase the circulation time in blood different approaches are classically carried out. These strategies are mainly based on the attachment of a pegylated moiety that extent the circulation time of the nanoparticle. However, in the case of Neridronate-IONP, the accumulation is produced very fast. The use of an aminobisphosphonate as biomolecule on the nanoparticles to target atheroma plaque is a new concept based on the calcium capabilities of these kinds of compounds. Its accumulation in the lesion area in less than an hr demonstrates the high affinity of Neridronate-IONP towards calcium contained in the atheroma plaque.
For visualization of atheroma plaque, many advanced imaging techniques are usually employed. Among them, positron emission tomography (PET) and MRI are the most standardized techniques. PET provides the best results in terms of functional information due to the high sensitivity and MRI the best results in anatomical information due to the high resolution. Although PET could be the ideal option to follow a synthetic probe, the resolution of this technique in small animals (~1 mm) restricts its use to visualize smaller calcifications in atherosclerotic lesions. MRI is an ideal alternative providing better resolution (~0.1 µm). The lower sensitivity of this technique not avoids the visualization of the contrast agent in the interest region and the better resolution allows identifying small calcifications. In addition, the results show that the combination of the unique fast accumulation of Neridronate-IONP with the high resolution of MRI is an ideal scenario for the detection of atheroma plaque in small animals.
The authors have nothing to disclose.
This study is supported by a grant from Comunidad de Madrid (S2010/BMD-2326, Inmunothercan-CM), by Fundacio La Marato de TV3 (70/C/2012) and by and by Spanish Economy Ministry (MAT2013-47303 P).
Microwave Explorer/Discover Hybrid-12 | CEM Corporation, USA | Any microwave for chemical synthesis can be used | |
Disposable PD-10 desalting columns | GE Healthcare life sciences | 17-0851-01 | Any size exclusion column will work |
Amicon®Ultra-0.5 ml | Merck Millipore Ltd | ||
Calibrated pH meter | SI analytics | 285105127 | |
Neodymium magnet | Aiman Gz | ND010B | |
Vortex Genius 3 | IKA | 3340000 | |
ZetaSizer Nano ZS | Malvern Instruments | ||
Standard (macro) cell Optical glass | Labbox | 11718 | |
Zetasizer nanoseries disponsable folded capillary cells DTS1070 | Malvern | ||
Bruker Minispec mq60 | Bruker |