Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Preparation of Reagents

1. Prepare 1 mM HEPES Buffer dissolving 23.8 mg of HEPES in 100 ml of distilled water. Adjust the pH to 7.
2. Prepare 10% NaHSO₃ dissolving 10 g of NaHSO₃ in 100 ml of distilled water. Stir the mixture for 15 min.
3. Prepare NaOH solution dissolving 1 g of NaOH in 100 ml of water. Stir for 10 min.
4. Prepare 10 mM Phosphate buffer dissolving 600 mg of NaH₂PO₄ in 1 L of water. Add carefully 0.34 ml of phosphoric acid and Stir for 30 min. Adjust the pH to 2.9 (acceptance range 2.7-3.0).
5. Prepare 10 mM Phosphate buffer dissolving 269 mg of NaH₂PO₄ and 1.09 g of Na₂HPO₄ to distilled water to make a volume of 1 L. Adjust the pH to 7.2.

2. Synthesis of Oleic Acid Coated Nanoparticles (OA-IONP)

1. In a microwave adapted flask add 0.5 g of Fe(acac)₃, 1.4 ml of oleic acid, 0.6 ml of oleylamine and 1.19 g of 1,2-hexadodecanediol. Add 10 ml of phenyl ether carefully through the flask wall using a graduated pipette.
2. Introduce the flask in the microwave reactor and start the microwave protocol.
   NOTE: Microwave software enables choosing the magnitude for different parameters like temperature, pressure, stirring speed, power and reaction time. Moreover, it has the possibility to load three different stages in the same protocol allowing tunable synthesis. Once the loaded synthetic protocol starts, microwave heats the sample as fast as possible (ramping process) and maintains it during the chosen reaction time (running process). Election of power determines the time of ramping.
3. Load a dynamic study in the microwave. The protocol contains three stages:
   1. Stage 1: set the temperature to 60 °C, time 2 min, pressure 250 psi and 150 W of power. Stir speed has to be in high position and max power in on.
   2. Stage 2: set the temperature to 200 °C, time 20 min, pressure 250 psi and 300 W of power. Stir speed has to be in high position and max power in on.
   3. Stage 3: set the temperature to 250 °C, time 10 min, pressure 250 psi and 300 W of power. Stir speed has to be in high position and max power in on.
4. After finishing the protocol, allow the flask to cool at room temperature.
   NOTE: Cooling down process can be done with or without gas stream. Both cases provide same results. Aggregates appear in the stir bar and in the wall of the flask, wash it with EtOH and put it onto the Erlenmeyer.
5. Transfer the reaction mixture to an Erlenmeyer using a glass pipette and add 10 ml of EtOH 98%. Put a Nd-Nb-B magnet below the flask, wait 5 min and remove the supernatant with a glass pipette.
6. Add 10 ml of EtOH, sonicate the sample at RT for 2 min and 40 kHz, put the sample on the magnet and eliminate the supernatant. Repeat this step at least three times.
7. Disperse oleic nanoparticles in 30 ml of CHCl₃ and sonicate at 40 kHz for 5 min at RT. Check the hydrodynamic size in the zetasizer as per manufacturer's instructions. Put 0.5 ml of OA-IONP in the glass cuvette and add 0.5 ml of CHCl₃. Acceptance range 7-10 nm expressed as Z-average size in intensity.
   NOTE: OA-IONP can be well-dispersed in hexane.

3. Synthesis of Azelaic Acid Nanoparticles (Azelaic Acid-IONP)

1. Dissolve 44.3 mg of KMnO₄ and 150.4 mg of BTACl in a mixture of H₂O:CHCl₃ (3:2 ml). Add the resultant solution to a 5 ml aliquot of OA-IONP in the microwave adapted flask.
2. Start the microwave protocol for Azelaic acid-IONP. Set temperature at 105 °C, time 9 min, pressure 250 psi and power at 300 W. Put 10 ml of phosphate buffer pH = 2.9 into the flask and repeat the microwave protocol. After cooling step, recover the nanoparticles using a magnet and eliminate the supernatant.
3. Add 5 ml of 10% NaHSO₃ to an Erlenmeyer flask, sonicate at 40 kHz for 2 min at 25 °C, collect the particles using a magnet and eliminate the supernatant. (The step is repeated 2 times.) Wash the nanoparticles three more times with 1% NaOH and finally re-disperse it in 5 ml of phosphate buffer pH = 7.2.
4. Check the hydrodynamic size and zeta potential. Put 0.7 ml of Azelaic-IONP into the disposable folded capillary cell and insert it onto the zetasizer.
   NOTE: Acceptance range for size 25-35 nm expressed as Z-average size in intensity. Acceptance range for Z-Potential -45 ± 5 mV. Larger nanoparticles (~70 nm) can be obtained with phosphate buffer pH >7 instead pH = 2.9 (ref Chem Eur J 2008).

4. Synthesis of Neridronate Nanoparticles (Neridronate-IONP)

1. Add 12 mg of EDC and 15 mg of Sulfo-NHS in an centrifuge with 2 ml aliquot of Azelaic-IONP. Put the mixture in a vortex at RT for 35 min.
2. Put a magnet below the centrifuge to destabilize the nanoparticles, aspirate the supernatant and wash the particles with 1.5 ml of HEPES 1 mM pH=7 buffer. (Repeat this step two times.) Afterwards, add 5 mg of neridronate and shake the mixture in a vortex for 2 hr.
3. Separate nanoparticles with a magnet and wash (3 x 2 ml) with 1 mM HEPES pH=7 buffer. Finally, disperse Neridronate-IONP in 2 ml of 1mM HEPES pH = 7 buffer.
4. Check the hydrodynamic size and zeta potential. Put 0.7 ml of Neridronate-IONP into the disposable folded capillary cell and insert it onto the zetasizer (see equipment setup).
   NOTE: Acceptance range for size 40-45 nm expressed as Z-average size in intensity. Acceptance range for Z-Potential -20 ± 5 mV.

5. In Vivo Detection of Atheroma Plaque in ApoE^-/- Mice by MRI

1. Preparation for the MRI Acquisition
   NOTE: Several extra systems for animal experimentation are needed. Thus, It will require:
   1. Use appropriate equipment to anesthetize the animals.
   2. Obtain a close circuit-circulating warm water system with an external warm air to keep the temperature of the animal stable.
      NOTE: In this case the MRI compatible monitoring and gating system registers the temperature of the animal inside the MRI magnet.
   3. Monitor the external temperature in the proximity of the animal, the body (rectal thermometer) temperature of the animal, the respiration sensor located under the body of the animal near the thorax using an integrated interface in the MRI console.
2. MRI Experiment
   1. Anesthetize the animals with vaporized isoflurane (2% for induction during two or three minutes and 1-1.5% for maintenance during the MRI experiment) with an 100% oxygen line.
   2. Place the animal in the center of the magnet with the help of a profile acquisition.
   3. After step 5.2.2, tune the RF coil to 300 MHz (7 T) resonance frequency and match the characteristic impedance of the coil to 50 Ohm for optimal signal reception.
      NOTE: Pay attention to the external wiring and connections going to the measurement system via the adaptor/splitter to each part of the transmitter coil individually (in our case it was a quadrature coil).
   4. After tuning and matching the coils, plug the coils into the scanner.
   5. For RF pulse calibration (shape and length) and center frequency adjustment perform both pulse calibration and center of frequency manually. Perform the 90° pulse calibration, coarse shimming (see below), center frequency and receiver gain adjustments manually.
   6. Perform exact position using a gradient echo (FLASH or GRE) localizer scan (3 plane scout acquisition: axial, coronal and sagittal, also called tripilot.
   7. Perform the magnet shimming to optimize the magnetic field homogeneity in the center of the magnet. Perform this step manually (see 5.2.3) using a one or single pulse FID sequence and adjust the first and second orders shims or any automatic shimming sequence included in the system
      NOTE: A well shimmed magnetic field is easily recognized and measured by the T2* (as bigger the better) or narrow FWHM of the spectra.
3. MRI Data Acquisition of the Plaque⁵
   1. Inject 100 µl (1 mg [Fe] ml^-1) of neridronate nanoparticles intravenously into tail vein and acquire images 1 hr post injection. Load the parameters for MRI acquisition of atherosclerotic plaque looking at the abdominal aorta (renal bifurcation).
   2. Put multi-slice, 10 to 20 slices in interleave mode to minimize artifacts.
   3. Acquire high-resolution fast spin echo T1-weighted MRI in coronal or axial view with the following parameters: FOV 60 x 30 mm (coronal), 30 x 30 mm (axial), slice thickness 0.8 mm (with the small gradient coil configuration this can be reduced to 0.6 mm), 400 msec TR, 8 msec TE, 256 x 256 acquisition and reconstruction matrix data, 6 (large gradient) to 8 (small gradient) signal averages for 5-8 min average acquisition times.
      NOTE: TE is especially critical and the presence of blood signal and flow and chemical shift artifacts that can limit the applications. In these cases flow insensitive fast spin echo T2-IR can help to reduce artifacts and the acquisition of complementary data in the exact same location can help to characterize the plaque. Additionally, a presaturation pulse can be used to reduce the fat tissue surrounding the arterial wall for better delineation of the outer boundary of the wall and chemical shift artifact reduction.
   4. Transfer the images using a standard format such as Dicom and view in appropriate software (e.g. Osirix Imaging Software or AMIDE: Medical Imaging Data Examiner)⁵. Quantify the contrast effect manually delimiting the vessel area, wall thickness, lumen area and plaque burden⁵.

Results

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 increa...

Discussion

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 nanopar...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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