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>

<ol>
	<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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superparamagnetic+nanosystems+based+on+iron+oxide+nanoparticles+for+biomedical+imaging.">Superparamagnetic nanosystems based on iron oxide nanoparticles for biomedical imaging.</a> Nanomed. 6, 519-528 (2011).</li><li>Carenza, 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=Rapid+synthesis+of+water-dispersible+superparamagnetic+iron+oxide+nanoparticles+by+a+microwave-assisted+route+for+safe+labeling+of+endothelial+progenitor+cells.">Rapid synthesis of water-dispersible superparamagnetic iron oxide nanoparticles by a microwave-assisted route for safe labeling of endothelial progenitor cells.</a> Acta Biomater. 10, 3775-3785 (2014).</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, 215602 (2012).</li><li>Gatti, D., Rossini, M., Viapiana, O., Idolazzi, L., Adami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+development+of+neridronate:+potential+for+new+applications.">Clinical development of neridronate: potential for new applications.</a> Ther. Clin. Risk Manag. 9, 139-147 (2013).</li><li>Drake, M. T., Clarke, B. L., Khosla, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphosphonates:+mechanism+of+action+and+role+in+clinical+practice.">Bisphosphonates: mechanism of action and role in clinical practice.</a> Mayo Clin Proc. 83, 1032-1045 (2008).</li><li>Devogelaer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+bone+diseases+with+bisphosphonates,+excluding+osteoporosis.">Treatment of bone diseases with bisphosphonates, excluding osteoporosis.</a> Curr. Opin. Rheumatol. 12, 331-335 (2000).</li><li>Pascu, O., 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=Surface+Reactivity+of+Iron+Oxide+Nanoparticles+by+Microwave-Assisted+Synthesis;+Comparison+with+the+Thermal+Decomposition+Route.">Surface Reactivity of Iron Oxide Nanoparticles by Microwave-Assisted Synthesis; Comparison with the Thermal Decomposition Route.</a> J. Phys. Chem. C. 116, 15108-15116 (2012).</li><li>Sun, S., Zeng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-Controlled+Synthesis+of+Magnetite+Nanoparticles.">Size-Controlled Synthesis of Magnetite Nanoparticles.</a> J. Am. Chem. Soc. 124, 8204-8205 (2002).</li><li>Hyeon, T., Lee, S. S., Park, J., Chung, Y., Na, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+highly+crystalline+and+monodisperse+maghemite+nanocrystallites+without+a+size-selection+process.">Synthesis of highly crystalline and monodisperse maghemite nanocrystallites without a size-selection process.</a> J. Am. Chem. Soc. 123, 12798-12801 (2001).</li><li>Herranz, F., Morales, M. P., Roca, A. G., Vilar, R., Ruiz-Cabello, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+aqueous+functionalization+of+superparamagnetic+Fe+2+O+3+nanoparticles.">A new method for the aqueous functionalization of superparamagnetic Fe 2 O 3 nanoparticles.</a> Contrast Media Mol. Imaging. 3, 215-222 (2008).</li><li>Herranz, F., Morales, M. P., Roca, A. G., Desco, M., Ruiz-Cabello, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+rapid+synthesis+of+water+stable+superparamagnetic+nanoparticles.">A new method for the rapid synthesis of water stable superparamagnetic nanoparticles.</a> Chem. Weinh. Bergstr. Ger. 14, 9126-9130 (2008).</li><li>Herranz, F., 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=Superparamagnetic+iron+oxide+nanoparticles+conjugated+to+a+grass+pollen+allergen+and+an+optical+probe.">Superparamagnetic iron oxide nanoparticles conjugated to a grass pollen allergen and an optical probe.</a> Contrast Media Mol. Imaging. 7, 435-439 (2012).</li></ol>

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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 nanopar...

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><tbody><tr><td>Microwave Explorer/Discover Hybrid-12</td><td>CEM Corporation, USA</td><td></td><td>Any microwave for chemical synthesis can be used</td></tr><tr><td>Disposable PD-10 desalting columns&amp;nbsp;</td><td>GE Healthcare life sciences</td><td>17-0851-01</td><td>Any size exclusion column will work</td></tr><tr><td>Amicon&amp;reg;Ultra-0.5 ml&amp;nbsp;</td><td>Merck Millipore Ltd</td><td></td><td></td></tr><tr><td>Calibrated pH meter&amp;nbsp;</td><td>SI analytics</td><td>285105127</td><td></td></tr><tr><td>Neodymium magnet&amp;nbsp;</td><td>Aiman Gz</td><td>ND010B</td><td></td></tr><tr><td>Vortex Genius 3&amp;nbsp;</td><td>IKA</td><td>3340000</td><td></td></tr><tr><td>ZetaSizer Nano ZS&amp;nbsp;</td><td>Malvern Instruments</td><td></td><td></td></tr><tr><td>Standard (macro) cell Optical glass&amp;nbsp;</td><td>Labbox</td><td>11718</td><td></td></tr><tr><td>Zetasizer nanoseries disponsable folded capillary cells DTS1070</td><td>Malvern</td><td></td><td></td></tr><tr><td>Bruker Minispec mq60</td><td>Bruker</td><td></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 incre...

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 ...

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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

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

Chemistry

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 ...

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse ...

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. ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in magnetic resonance imaging (MRI). While iron oxide nanoparticles provide constant negative contrast on MRI over typical experimental timeframes, MnO generates switchable positive contrast on MRI through dissolution of MnO to Mn2+ at low pH within cell endosomes to &#8216;turn ON&#8217; MRI contrast. This protocol describes a one-pot synthesis of MnO nanoparticles formed by thermal decomposition of manganese(II) acetylacetonate in oleylamine and dibenzyl ether. Although running the synthesis of MnO nanoparticles is simple, the initial experimental setup can be difficult to reproduce if detailed instructions are not provided. Thus, the glassware and tubing assembly is first thoroughly described to allow other investigators to easily reproduce the setup. The synthesis method incorporates a temperature controller to achieve automated and precise manipulation of the desired temperature profile, which will impact resulting nanoparticle size and chemistry. The thermal decomposition protocol can be readily adapted to generate other metal oxide nanoparticles (e.g., iron oxide) and to include alternative organic solvents and stabilizers (e.g., oleic acid). In addition, the ratio of organic solvent to stabilizer can be changed to further impact nanoparticle properties, which is shown herein. Synthesized MnO nanoparticles are characterized for morphology, size, bulk composition, and surface composition through transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy, respectively. The MnO nanoparticles synthesized by this method will be hydrophobic and must be further manipulated through ligand exchange, polymeric encapsulation, or lipid capping to incorporate hydrophilic groups for interaction with biological fluids and tissues.

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of ManganeseII Acetylacetonate

This protocol details a facile, one-pot synthesis of manganese oxide (MnO) nanoparticles by thermal decomposition of manganese(II) acetylacetonate in the presence of oleylamine and dibenzyl ether. MnO nanoparticles have been utilized in diverse applications including magnetic resonance imaging, biosensing, catalysis, batteries, and waste water treatment.

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

Biology

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

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

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Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

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

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection