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).

1. Preparation of Reagents
<ol>
	<li>Prepare 1 mM HEPES Buffer dissolving 23.8 mg of HEPES in 100 ml of distilled water. Adjust the pH to 7.</li>
	<li>Prepare 10% NaHSO3 dissolving 10 g of NaHSO3 in 100 ml of distilled water. Stir the mixture for 15 min.</li>
	<li>Prepare NaOH solution dissolving 1 g of NaOH in 100 ml of water. Stir for 10 min.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
2. Synthesis of Oleic Acid Coated Nanoparticles (OA-IONP)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Load a dynamic study in the microwave. The protocol contains three stages:
	<ol>
		<li>Stage 1: set the temperature to 60 &#176;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.</li>
		<li>Stage 2: set the temperature to 200 &#176;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.</li>
		<li>Stage 3: set the temperature to 250 &#176;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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;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.</li>
</ol>
3. Synthesis of Azelaic Acid Nanoparticles (Azelaic Acid-IONP)
<ol>
	<li>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.</li>
	<li>Start the microwave protocol for Azelaic acid-IONP. Set temperature at 105 &#176;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.</li>
	<li>Add 5 ml of 10% NaHSO3 to an Erlenmeyer flask, sonicate at 40 kHz for 2 min at 25 &#176;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.</li>
	<li>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 &#177; 5 mV. Larger nanoparticles (~70 nm) can be obtained with phosphate buffer pH &#62;7 instead pH = 2.9 (ref Chem Eur J 2008).</li>
</ol>
4. Synthesis of Neridronate Nanoparticles (Neridronate-IONP)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#177; 5 mV.</li>
</ol>
5. In Vivo Detection of Atheroma Plaque in ApoE-/- Mice by MRI
<ol>
	<li>Preparation for the MRI Acquisition 
	NOTE: Several extra systems for animal experimentation are needed. Thus, It will require:
	<ol>
		<li>Use appropriate equipment to anesthetize the animals.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>MRI Experiment
	<ol>
		<li>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.</li>
		<li>Place the animal in the center of the magnet with the help of a profile acquisition.</li>
		<li>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).</li>
		<li>After tuning and matching the coils, plug the coils into the scanner.</li>
		<li>For RF pulse calibration (shape and length) and center frequency adjustment perform both pulse calibration and center of frequency manually. Perform the 90&#176; pulse calibration, coarse shimming (see below), center frequency and receiver gain adjustments manually.</li>
		<li>Perform exact position using a gradient echo (FLASH or GRE) localizer scan (3 plane scout acquisition: axial, coronal and sagittal, also called tripilot.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>MRI Data Acquisition of the Plaque5
	<ol>
		<li>Inject 100 &#181;l (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).</li>
		<li>Put multi-slice, 10 to 20 slices in interleave mode to minimize artifacts.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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	<li>Patel, D. N., Bailey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+in+cardiovascular+medicine.">Nanotechnology in cardiovascular medicine.</a> Catheter. Cardiovasc. Interv. Off. J. Soc. Card. Angiogr. Interv. 69, 643-654 (2007).</li><li>Zhao, X., Zhao, H., Chen, Z., Lan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+superparamagnetic+iron+oxide+nanoparticles+for+magnetic+resonance+imaging+contrast+agent.">Ultrasmall superparamagnetic iron oxide nanoparticles for magnetic resonance imaging contrast agent.</a> J. Nanosci. Nanotechnol. 14, 210-220 (2014).</li><li>Lee, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+nanoparticles+for+multimodal+imaging+and+theragnosis.">Multifunctional nanoparticles for multimodal imaging and theragnosis.</a> Chem. Soc. Rev. 41, 2656 (2012).</li><li>Osborne, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+microwave-assisted+synthesis+of+dextran-coated+iron+oxide+nanoparticles+for+magnetic+resonance+imaging.">Rapid microwave-assisted synthesis of dextran-coated iron oxide nanoparticles for magnetic resonance imaging.</a> Nanotechnology. 23, (2012).</li><li>Pellico, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-driven+synthesis+of+bisphosphonate+nanoparticles+allows+in+vivo+visualisation+of+atherosclerotic+plaque.">Microwave-driven synthesis of bisphosphonate nanoparticles allows in vivo visualisation of atherosclerotic plaque.</a> RSC Adv. 5, 1661-1665 (2015).</li><li>Lin, M. M., Kim, D. K., El Haj, A. J., Dobson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+superparamagnetic+iron+oxide+nanoparticles+(SPIONS)+for+translation+to+clinical+applications.">Development of superparamagnetic iron oxide nanoparticles (SPIONS) for translation to clinical applications.</a> IEEE Trans. Nanobioscience. 7, 298-305 (2008).</li><li>Gupta, A. K., Gupta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+surface+engineering+of+iron+oxide+nanoparticles+for+biomedical+applications.">Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications.</a> Biomaterials. 26, 3995-4021 (2005).</li><li>Liu, F., Laurent, S., Fattahi, H., Vander Elst, L., Muller, R. 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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 &#60;50 nm) using pH = 2.9 to larger hydrodynamic size (Dh &#62;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 &#181;m). 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.

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&#160; 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&#39;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 &#177; 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 &#177; 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 &#177; 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.

<table border="0" cellpadding="0" cellspacing="0" width="1107">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Microwave Explorer/Discover Hybrid-12</td>
			<td style="width:176px;">CEM Corporation, USA</td>
			<td style="width:225px;"></td>
			<td style="width:311px;">Any microwave for chemical synthesis can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable PD-10 desalting columns&#160;</td>
			<td>GE Healthcare life sciences</td>
			<td>17-0851-01</td>
			<td>Any size exclusion column will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon&#174;Ultra-0.5 ml&#160;</td>
			<td>Merck Millipore Ltd</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calibrated pH meter&#160;</td>
			<td>SI analytics</td>
			<td>285105127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neodymium magnet&#160;</td>
			<td>Aiman Gz</td>
			<td>ND010B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genius 3&#160;</td>
			<td>IKA</td>
			<td>3340000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZetaSizer Nano ZS&#160;</td>
			<td>Malvern Instruments</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard (macro) cell Optical glass&#160;</td>
			<td>Labbox</td>
			<td>11718</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zetasizer nanoseries disponsable folded capillary cells DTS1070</td>
			<td>Malvern</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:395px;">Bruker Minispec mq60</td>
			<td style="width:176px;">Bruker</td>
			<td style="width:225px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

microwave-driven synthesis of iron oxide nanoparticles for fast detection of atherosclerosis

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.

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 &#60;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&#160;&#181;l (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 &#60;0.05, Figure 2b).
In addition, signal in the liver was monitored in mice after injection of 100 &#181;l 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).
<img alt="Scheme 1" src="/files/ftp_upload/53472/53472sch1highres.jpg" width="700" />
Scheme 1: Synthetic steps followed in the protocol and basic characterization at each point by DLS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53472/53472sch1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/53472/53472fig1highres.jpg" width="700" /> 
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). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53472/53472fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53472/53472fig2highres.jpg" width="700" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53472/53472fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis at JoVE.com

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

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 ...

microwave-driven-synthesis-iron-oxide-nanoparticles-for-fast

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.4K Views.  Centro Nacional de Investigaciones Cardiovasculares Carlos III and CIBER de Enfermedades Respiratorias. 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.

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

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...