Name: using magnetometry to monitor cellular incorporation and subsequent biodegradation of chemically synthetized iron oxide nanoparticles
Uploaded: 2021-02-27T13:30:00
Description: Watch this Scientific Journal Video about Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles at JoVE.com

This work was supported by the European Union (ERC-2014-CoG project MaTissE #648779). The authors would like to acknowledge&#160;the CNanoMat physico-chemical characterizations platform of University Paris 13.

1. Synthesis of magnetic nanoparticles
<ol>
	<li>Core synthesis &#8211; microwave-assisted
	<ol>
		<li>Dissolve 400 mg of iron(III) acetylacetonate (&#62;99.9%) in 10 mL of benzyl alcohol (BA, 99.8%) within a 30 mL monowave glass vial.</li>
		<li>Increase the temperature of the suspension from 25 to 250 &#176;C in 20 min (at a rate of 11.25 &#176;C/min) and maintain it at 250 &#176;C for 30 min using a microwave reactor.</li>
		<li>Transfer the resulting nanoparticles suspended in benzyl alcohol to a glass vial and sep...

<ol>
	<li>Azevedo-Pereira, R. L., 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+as+a+tool+to+track+mouse+neural+stem+cells+in+vivo.">Superparamagnetic iron oxide nanoparticles as a tool to track mouse neural stem cells in vivo.</a> Molecular Biology Reports. 46 (1), 191-198 (2019).</li><li>Fayol, D., Frasca, G., Le Visage, C., Gazeau, F., Luciani, N., Wilhelm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+magnetic+forces+to+promote+stem+cell+aggregation+during+differentiation,+and+cartilage+tissue+modeling.">Use of magnetic forces to promote stem cell aggregation during differentiation, and cartilage tissue modeling.</a> Advanced Materials. 25 (18), 2611-2616 (2013).</li><li>Kim, J. 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=High-throughput+generation+of+spheroids+using+magnetic+nanoparticles+for+three-dimensional+cell+culture.">High-throughput generation of spheroids using magnetic nanoparticles for three-dimensional cell culture.</a> Biomaterials. 34 (34), 8555-8563 (2013).</li><li>Yamamoto, Y., 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=Preparation+of+artificial+skeletal+muscle+tissues+by+a+magnetic+force-based+tissue+engineering+technique.">Preparation of artificial skeletal muscle tissues by a magnetic force-based tissue engineering technique.</a> Journal of Bioscience and Bioengineering. 108 (6), 538-543 (2009).</li><li>Gon&#231;alves, A. I., Rodrigues, M. T., Gomes, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+magnetic+cell+sheet+patches+for+advanced+strategies+in+tendon+regeneration.">Tissue-engineered magnetic cell sheet patches for advanced strategies in tendon regeneration.</a> Acta Biomaterialia. 63, 110-122 (2017).</li><li>Du, V., 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=A+3D+magnetic+tissue+stretcher+for+remote+mechanical+control+of+embryonic+stem+cell+differentiation.">A 3D magnetic tissue stretcher for remote mechanical control of embryonic stem cell differentiation.</a> Nature Communications. 8 (1), 400 (2017).</li><li>Amiri, M., Salavati-Niasari, M., Pardakhty, A., Ahmadi, M., Akbari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine:+A+novel+green+precursor+for+synthesis+of+magnetic+CoFe2O4+nanoparticles+and+pH-sensitive+magnetic+alginate+beads+for+drug+delivery.">Caffeine: A novel green precursor for synthesis of magnetic CoFe2O4 nanoparticles and pH-sensitive magnetic alginate beads for drug delivery.</a> Materials Science and Engineering: C. 76, 1085-1093 (2017).</li><li>Vangijzegem, T., Stanicki, D., Laurent, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+iron+oxide+nanoparticles+for+drug+delivery:+applications+and+characteristics.">Magnetic iron oxide nanoparticles for drug delivery: applications and characteristics.</a> Expert Opinion on Drug Delivery. 16 (1), 69-78 (2019).</li><li>Cazares-Cortes, 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=Recent+insights+in+magnetic+hyperthermia:+From+the+"hot-spot"+effect+for+local+delivery+to+combined+magneto-photo-thermia+using+magneto-plasmonic+hybrids.">Recent insights in magnetic hyperthermia: From the "hot-spot" effect for local delivery to combined magneto-photo-thermia using magneto-plasmonic hybrids.</a> Advanced Drug Delivery Reviews. 138, 233-246 (2019).</li><li>Espinosa, 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=Magnetic+(Hyper)Thermia+or+Photothermia?+Progressive+Comparison+of+Iron+Oxide+and+Gold+Nanoparticles+Heating+in+Water,+in+Cells,+and+in+vivo.">Magnetic (Hyper)Thermia or Photothermia? Progressive Comparison of Iron Oxide and Gold Nanoparticles Heating in Water, in Cells, and in vivo.</a> Advanced Functional Materials. 28 (37), 1803660 (2018).</li><li>Plan Sangnier, 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=Targeted+thermal+therapy+with+genetically+engineered+magnetite+magnetosomes@RGD:+Photothermia+is+far+more+efficient+than+magnetic+hyperthermia.">Targeted thermal therapy with genetically engineered magnetite magnetosomes@RGD: Photothermia is far more efficient than magnetic hyperthermia.</a> Journal of Controlled Release. 279, 271-281 (2018).</li><li>Pham, B. T. T., 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=Biodistribution+and+Clearance+of+Stable+Superparamagnetic+Maghemite+Iron+Oxide+Nanoparticles+in+Mice+Following+Intraperitoneal+Administration.">Biodistribution and Clearance of Stable Superparamagnetic Maghemite Iron Oxide Nanoparticles in Mice Following Intraperitoneal Administration.</a> International Journal of Molecular Sciences. 19 (1), (2018).</li><li>Kolosnjaj-Tabi, 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=Biotransformations+of+magnetic+nanoparticles+in+the+body.">Biotransformations of magnetic nanoparticles in the body.</a> Nano Today. 11 (3), 280-284 (2016).</li><li>Bargheer, D., 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=The+distribution+and+degradation+of+radiolabeled+superparamagnetic+iron+oxide+nanoparticles+and+quantum+dots+in+mice.">The distribution and degradation of radiolabeled superparamagnetic iron oxide nanoparticles and quantum dots in mice.</a> Beilstein Journal of Nanotechnology. 6, 111-123 (2015).</li><li>Freund, B., 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=A+simple+and+widely+applicable+method+to+59Fe-radiolabel+monodisperse+superparamagnetic+iron+oxide+nanoparticles+for+in+vivo+quantification+studies.">A simple and widely applicable method to 59Fe-radiolabel monodisperse superparamagnetic iron oxide nanoparticles for in vivo quantification studies.</a> ACS Nano. 6 (8), 7318-7325 (2012).</li><li>Singh, S. P., Rahman, M. F., Murty, U. S. N., Mahboob, M., Grover, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+genotoxicity+and+tissue+distribution+of+nano+and+micron+sized+iron+oxide+in+rats+after+acute+oral+treatment.">Comparative study of genotoxicity and tissue distribution of nano and micron sized iron oxide in rats after acute oral treatment.</a> Toxicology and Applied Pharmacology. 266 (1), 56-66 (2013).</li><li>Levy, M., 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=Long+term+in+vivo+biotransformation+of+iron+oxide+nanoparticles.">Long term in vivo biotransformation of iron oxide nanoparticles.</a> Biomaterials. 32 (16), 3988-3999 (2011).</li><li>Briley-Saebo, K., 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=Hepatic+cellular+distribution+and+degradation+of+iron+oxide+nanoparticles+following+single+intravenous+injection+in+rats:+implications+for+magnetic+resonance+imaging.">Hepatic cellular distribution and degradation of iron oxide nanoparticles following single intravenous injection in rats: implications for magnetic resonance imaging.</a> Cell and Tissue Research. 316 (3), 315-323 (2004).</li><li>Gu, L., Fang, R. H., Sailor, M. J., Park, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Clearance+and+Toxicity+of+Monodisperse+Iron+Oxide+Nanocrystals.">In vivo Clearance and Toxicity of Monodisperse Iron Oxide Nanocrystals.</a> ACS Nano. 6 (6), 4947-4954 (2012).</li><li>Sangnier, A. P., 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=Impact+of+magnetic+nanoparticle+surface+coating+on+their+long-term+intracellular+biodegradation+in+stem+cells.">Impact of magnetic nanoparticle surface coating on their long-term intracellular biodegradation in stem cells.</a> Nanoscale. , (2019).</li><li>Mazuel, 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=Magneto-Thermal+Metrics+Can+Mirror+the+Long-Term+Intracellular+Fate+of+Magneto-Plasmonic+Nanohybrids+and+Reveal+the+Remarkable+Shielding+Effect+of+Gold.">Magneto-Thermal Metrics Can Mirror the Long-Term Intracellular Fate of Magneto-Plasmonic Nanohybrids and Reveal the Remarkable Shielding Effect of Gold.</a> Advanced Functional Materials. 27 (9), 1605997 (2017).</li><li>Van de Walle, 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=Biosynthesis+of+magnetic+nanoparticles+from+nano-degradation+products+revealed+in+human+stem+cells.">Biosynthesis of magnetic nanoparticles from nano-degradation products revealed in human stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (10), 4044-4053 (2019).</li><li>Mazuel, 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=Massive+Intracellular+Biodegradation+of+Iron+Oxide+Nanoparticles+Evidenced+Magnetically+at+Single-Endosome+and+Tissue+Levels.">Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single-Endosome and Tissue Levels.</a> ACS Nano. 10 (8), 7627-7638 (2016).</li><li>Bee, A., Massart, R., Neveu, S. <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+very+fine+maghemite+particles.">Synthesis of very fine maghemite particles.</a> Journal of Magnetism and Magnetic Materials. 149 (1), 6-9 (1995).</li><li>Feldmann, C., Jungk, H. 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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> Journal of the American Chemical Society. 123 (51), 12798-12801 (2001).</li><li>Pinna, N., Grancharov, S., Beato, P., Bonville, P., Antonietti, M., Niederberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetite+Nanocrystals:+Nonaqueous+Synthesis,+Characterization,+and+Solubility.">Magnetite Nanocrystals: Nonaqueous Synthesis, Characterization, and Solubility.</a> Chemistry of Materials. 17 (11), 3044-3049 (2005).</li><li>Richard, S., 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=USPIO+size+control+through+microwave+nonaqueous+sol-gel+method+for+neoangiogenesis+T2+MRI+contrast+agent.">USPIO size control through microwave nonaqueous sol-gel method for neoangiogenesis T2 MRI contrast agent.</a> Nanomedicine. 11 (21), 2769-2797 (2016).</li><li>Ngo, A. T., Pileni, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assemblies+of+Ferrite+Nanocrystals:+Partial+Orientation+of+the+Easy+Magnetic+Axes.">Assemblies of Ferrite Nanocrystals: Partial Orientation of the Easy Magnetic Axes.</a> The Journal of Physical Chemistry B. 105 (1), 53-58 (2001).</li><li>Li, L., 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=Effect+of+synthesis+conditions+on+the+properties+of+citric-acid+coated+iron+oxide+nanoparticles.">Effect of synthesis conditions on the properties of citric-acid coated iron oxide nanoparticles.</a> Microelectronic Engineering. 110, 329-334 (2013).</li><li>Sun, X., 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=Tracking+stem+cells+and+macrophages+with+gold+and+iron+oxide+nanoparticles+-+The+choice+of+the+best+suited+particles.">Tracking stem cells and macrophages with gold and iron oxide nanoparticles - The choice of the best suited particles.</a> Applied Materials Today. 15, 267-279 (2019).</li><li>Buchner, M., H&#246;fler, K., Henne, B., Ney, V., Ney, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tutorial:+Basic+principles,+limits+of+detection,+and+pitfalls+of+highly+sensitive+SQUID+magnetometry+for+nanomagnetism+and+spintronics.">Tutorial: Basic principles, limits of detection, and pitfalls of highly sensitive SQUID magnetometry for nanomagnetism and spintronics.</a> Journal of Applied Physics. 124 (16), 161101 (2018).</li><li>Wilhelm, C., Gazeau, F., Bacri, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetophoresis+and+ferromagnetic+resonance+of+magnetically+labeled+cells.">Magnetophoresis and ferromagnetic resonance of magnetically labeled cells.</a> European Biophysics Journal. 31 (2), 118-125 (2002).</li><li>Jing, Y., 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=Quantitative+intracellular+magnetic+nanoparticle+uptake+measured+by+live+cell+magnetophoresis.">Quantitative intracellular magnetic nanoparticle uptake measured by live cell magnetophoresis.</a> The FASEB Journal. 22 (12), 4239-4247 (2008).</li><li>Van de Walle, 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=Real-time+in+situ+magnetic+measurement+of+the+intracellular+biodegradation+of+iron+oxide+nanoparticles+in+a+stem+cell-spheroid+tissue+model.">Real-time in situ magnetic measurement of the intracellular biodegradation of iron oxide nanoparticles in a stem cell-spheroid tissue model.</a> Nano Research. , (2020).</li></ol>

Using a fast and efficient microwave-based synthesis, magnetic nanoparticles can easily be synthesized, with controlled size, and further coated with given molecules. A critical step is to stock the iron salt and the benzyl alcohol under vacuum to keep a small dispersion in size. The benzyl alcohol acts as both as solvent and ligand at the same time allowing to directly obtain calibrated bare iron oxide without the need of additional ligands. After nanoparticles transfer in water the bare magnetic nanoparticles can be ea...

There is increased interest in the magnetic features of iron oxide nanoparticles for a wide range of biomedical applications. Their response to magnetic resonance makes them reliable contrast agents for magnetic resonance imaging (MRI), an advantage in regenerative medicine where cells labeled with magnetic nanoparticles can be tracked in vivo following implantation1. Using magnetic fields, cells can also be guided at a distance; this way, cellular spheroids2,3, rings4, or sheets5 can be engineered magnetically and also remotely stimulated6, an asset in the development of scaffold-free tissues. The range of possibilities for these nanoparticles also includes drug delivery systems7,8 and magnetic and photoinduced hyperthermal treatment to kill cancerous cells9,10,11. For all these applications, the nanoparticles are integrated in the biological environment either by intravenous injection or via direct internalization in cells and are then left within, which brings into question their intracellular fate.
In vivo analyses conveyed a general understanding of the nanoparticles&#8217; fate in the organism: upon injection in the blood stream, they are first captured mostly by the macrophages of the liver (Kupffer cells), spleen and bone marrow, are progressively degraded, and join the iron pool of the organism12,13,14,15,16,17,18,19. Qualitative observations are only possible due to the circulation of the nanoparticles throughout the organism. Typically, transmission electronic microscopy (TEM) can be used to directly observe the nanoparticles and the presence of iron in the organs can be determined via dosage. More recently, their fate has been assessed directly on a pool of cells, meaning in close circuit with no iron escape, allowing a quantitative measurement of their biotransformations at the cell-level20,21,22. Such measurements are possible via the analysis of the magnetic properties of the nanoparticles that are tightly linked to their structural integrity. Vibrating sample magnetometry (VSM) is a technique where the sample is vibrated periodically so that the coil-measurement of the flux induced provides the magnetic moment of the sample at the applied magnetic field. Such synchronous detection allows for a rapid measurement, which is an asset for determining the magnetic moments of a large number of samples20,21,22,23. The macroscopic magnetic signature retrieved by VSM then gives a quantitative overview of the entire biological sample directly correlated to the nanoparticles&#39; size and structure. In particular, it provides the magnetic moment at saturation (expressed in emu) of the samples, which is a direct quantification of the number of magnetic nanoparticles present in the sample, respectively to their specific magnetic properties.
It has been shown that the intracellular processing of magnetic nanoparticles is tightly linked to their structural features20. These features can be controlled via optimal synthesis protocols. Each protocol presents advantages and limitations. Iron oxide nanoparticles are commonly synthesized in aqueous solutions via coprecipitation of iron ions24. To overcome the limitations of nanoparticles size polydispersity, other synthesis methods such as polyol-mediated sol-gel methods have been developed25. Nonaqueous approaches by thermal decomposition leads to the production of very well-calibrated iron oxide nanoparticles26. However, the use of massive amounts of surfactants like oleylamine or oleic acid complicates their functionalization and water transfer for biomedical applications. For this reason, we synthesize such magnetic nanoparticles through a nonaqueous sol gel route leading to high crystallinity, purity and reproducibility27. This protocol produces well-controlled size nanoparticles that can be tuned through temperature variation28. Nevertheless, the microwave-assisted non-aqueous sol-gel route has an upper size limit of the obtained nanoparticles of around 12 nm. This procedure would not be adapted for applications using ferromagnetic particles at room temperature. In addition to the core synthesis, another main feature to be considered is the coating. Lying at the surface of the nanoparticle, the coating acts as an anchoring molecule, helping the targeted internalization of the nanoparticles, or it can protect the nanoparticle from degradation. Since benzyl alcohol acts as an oxygen source and a ligand at the same time, bare nanoparticles are produced without the need for additional surfactants or ligands. The nanoparticles are then easily surface functionalized after synthesis without a surfactant exchange process.
Herein, two types of nanoparticles are assessed that possess the same core and differ in the coating. The core is synthesized using a fast and highly efficient microwave based technique. The two coatings compared consist of citric acid, one of the most used as coating agent in biomedical applications29,30, and polyacrylic acid (PAA), a polymeric coating with a high number of chelating functions. VSM magnetometry measurements are then used first to quantify the nanoparticle uptake by the cells, and then as a direct assessment of the nanoparticle structural integrity upon internalization in stem cells. Results demonstrate that the incubation concentration impacts nanoparticle uptake and that the coating influences their degradation, with the large number of anchoring molecules of PAA protecting the core from degradation.

<table>
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl alcohol for synthesis</td>
			<td>Sigma Aldrich</td>
			<td>8.22259</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Prepare a 1 mM stock solution diluted in Ethanol 100% and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Dichloromethane &#8805;99% stabilised, GPR RECTAPUR</td>
			<td>VWR Chemicals</td>
			<td>23367</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with Glutamax I</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>No sodium pyruvate, no HEPES</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR</td>
			<td>20821.310</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution 10% neutral buffered</td>
			<td>Sigma</td>
			<td>HT5012</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 1.0N Standardized Solution</td>
			<td>Alfa Aesar</td>
			<td>35640</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron(III) acetylacetonate (&#62; 99.9%)</td>
			<td>Sigma Aldrich</td>
			<td>517003</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Premix Universal Culture Supplement (20x)</td>
			<td>Corning</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphate</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td>Prepare a fresh concentrated solution (25 mM) diluted in distilled water</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma</td>
			<td>P5607</td>
			<td>Prepare a 175 mM stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Mesenchymal Stem Cell (MSC)</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>Monowave glass vial</td>
			<td>Anton Paar</td>
			<td>82723_us</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave reactor</td>
			<td>Anton Paar</td>
			<td>Monowave 300</td>
			<td></td>
		</tr>
		<tr>
			<td>MSCGM BulletKit medium</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td>For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium</td>
		</tr>
		<tr>
			<td>PBS w/o CaCl2 w/o MgCl2</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin (10.000U/mL)/Streptomicin (10.000&#181;g/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(acrylic acid, sodium salt)</td>
			<td>Sigma Aldrich</td>
			<td>416010</td>
			<td>MW = 1200 g/mol</td>
		</tr>
		<tr>
			<td>RPMI medium 1640, no Glutamine</td>
			<td>Life Technologies</td>
			<td>31870-025</td>
			<td>No sodium pyruvate, no HEPES</td>
		</tr>
		<tr>
			<td>Sodium hydroxide, 1.0N Standardized Solution</td>
			<td>Alfa Aesar</td>
			<td>35629</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate solution 100mM</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile conical centrifuge tube</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>15 mL tubes</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-sodium citrate</td>
			<td>VWR</td>
			<td>33615.268</td>
			<td>Prepare a 1 M stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Tri-Sodium Citrate Dihydrate, Certified AR for Analysis</td>
			<td>Sigma Aldrich</td>
			<td>10396430</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra centrifugal filter</td>
			<td>Amicon</td>
			<td>AC S510024</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the microwave-assisted synthesis, magnetic nanoparticles with a monodisperse 8.8 &#177; 2.5 nm core size are produced and coated with either citrate or PAA (Figure 1A). Stem cells are then incubated with these nanoparticles dispersed in culture medium at a given concentration for 30 minutes, resulting in their endocytosis and confinement within the cellular endosomes (Figure 1B). The magnetic stem cells are then suspended in medium, centrifuged, and the ce...

Watch this Scientific Journal Video about Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles at JoVE.com

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

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 ...

Research

JoVE Journal

Bioengineering

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

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 ...

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 ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

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 ...

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 ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...