Name: microwave-driven synthesis of iron oxide nanoparticles for fast detection of atherosclerosis
Uploaded: 2016-03-22T14:00:00
Description: Watch this Scientific Journal Video about Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis at JoVE.com

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 S...

<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. 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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. 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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 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 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 ...

The overall goal of this protocol is the use of microwave technology for the rapid synthesis and functionalization of iron oxide nanoparticles with a bisphosphonate moiety for fast detection by MRI of atherosclerosis in mice models. The use of microwave technology in the synthesis and functionalization of iron oxide nanoparticles is still limited. However, we demonstrate how high quality nanoparticles and extremely fast protocols can be developed.The main advantage of this technique is that in extremely short times, high quality functionalized nanoparticles can be produced and used for catheterization of atherosclerosis plaque targeting microcalcifications. To begin this procedure, add 0.5 grams of iron 3-acetylacetenate, 1.4 milliliters of oleic acid, 0.6 milliliters of oleylamine, and 1.19 grams of 1, 2-Hexadodecanediol to a microwave-adapted flask. Add 10 milliliters of phenyl ether carefully down the flask wall using a graduated pipette.Following this, place the flask in a microwave reactor and start the microwave protocol by loading a dynamic study in the microwave. After the reaction is complete, and the flask has cooled to room temperature, transfer the reaction mixture to an Erlenmeyer flask using a glass pipette and add 10 milliliters of 98 percent ethanol. Place the flask on top of a neodymium niobium boron magnet and remove the supernatant with a glass pipette after 5 minutes.Next, add 10 milliliters of ethanol to the flask. Sonicate the sample at 40 kilohertz for two minutes at room temperature. When finished, place the sample on the magnet and remove the supernatant.Disperse the oleic acid coated nanoparticles in 30 milliliters of chloroform and sonicate at 40 kilohertz for five minutes at room temperature. Following sonication, transfer 0.5 milliliters of the nanoparticles in a glass cuvette and add 0.5 milliliters of chloroform. Check the hydrodynamic size in a zetasizer per the manufacturer's instructions.At this point, dissolve 44.3 milligrams of potassium permanganate and 150.4 milligrams of benzoyl trimethyl ammonium chloride in a three to two mixture of water and chloroform. Add the resultant solution to a 5 milliliter Aliquat of the oleic-acid coated nanoparticles in a microwave-adapted flask. Start the microwave protocol for azelaic acid nanoparticles by setting the temperature to 105 degrees Celsius, the time to nine minutes, the pressure to 250 psi, and the power to 300 watts.Following this, add 10 milliliters of pH 2.9 phosphate buffer to the flask. After repeating the microwave protocol, and removing the supernatant, add 10 milliliters of water to the flask and transfer the mixture to an Erlenmeyer flask. The most critical step is purification of azelaic acid nanoparticles.Next, add 5 milliliters of 10 percent sodium bisulphate to the Erlenmeyer flask and sonicate at 40 kilohertz for two minutes at 25 degrees Celsius. After collecting the particles, and removing the supernatant, wash the nanoparticles three more times with 1 percent sodium hydroxide. Redisperse the particles in 5 milliliters of pH 7.2 phosphate buffer.To check the hydrodynamic size and zeta potential, transfer 0.7 milliliters of the azelaic acid nanoparticles to a disposable, folded capillary cell and place it in the zetasizer for analysis. Add 12 milligrams of EDC and 15 milligrams of sulfo-NHS in a centrifuge tube with a 2 milliliter Aliquat of the azelaic acid nanoparticles. Vortex the mixture at room temperature for 35 minutes.After placing a magnet below the centrifuge tube to destabilize the nanoparticles, aspirate the supernatant and wash the particles with 1.5 milliliters of 1 millimolar hepes buffer. Then, add 5 milligrams of neridronate and vortex the mixture for two hours. Separate the nanoparticles with a magnet and wash with 1 millimolar hepes buffer.Finally, disperse the neridronate nanoparticles in 2 milliliters of 1 millimolar hepes buffer. To check the hydrodynamic size and zeta potential, add 0.7 milliliters of the neridronate nanoparticles to a disposable folded capillary cell and place it in the zetsizer for analysis. All nanoparticles presented small hydrodynamic size in a very narrow size distribution.The particles present excellent crystallinity as shown in the TEM images. Additionally, the same results for hydrodynamic size and distribution were obtained after four repetitions of the synthesis, demonstrating one of the most important aspects of the microwave approach, its reproducability. The calcium ion binding properties due to bisphosphonates present in the neridronate nanoparticles were checked by relaxometry and demonstrated that T2 relaxation time increments linearly with the calcium ion amount and incubation time due to the formation of nanoparticle clusters whereas nanoparticles without calcium ion remained stable.In vivo MRI experiments were performed in 48-week old apoe knockout mice and at one hour post injection, the signal from the plaque was hypointense in comparison to the basal images. Additionally, the plaque to muscle ratio was significantly different between the basal and one hour post injection images. The signal in the liver was also monitored after injection of the neridronate nanoparticles which were completely cleared from circulation after 20 minutes, confirming the selective accumulation of these nanoparticles towards atherosclerotic plaque.Ex vivo imaging and histology were performed and imaging of aortas, with and without nanoparticles, showed differences in the signal intensity in agreement with the in vivo experiments. Once mastered, the microwave technology allows for the synthesis characterization and plaque detection in a total time of 3 hours if properly performed. Another advantage of this method is that, with a small modification other imaging techniques, like thermography can be performed, in order to answer additional questions like quantification of atherosclerosis.After its development, this technique paved the way for researchers in the field of cardiovascular imaging to explore the early diagnosis of atherosclerosis with an experimental setup that can be easily adapted to clinical needs. After watching this video, you should have a good understanding of how to produce hydrophilic iron oxide nanoparticles with microwave and its application in cardiovascular imaging. Don't forget that working with microwave ovens can be hazardous depending on the oven used, and precautions such as pressure control of the cistern should always be taken while performing this procedure.

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