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 separate the nanoparticles using a neodymium magnet for 30 min.</li>
		<li>Wash the precipitate in the previous 100 mL glass vial with 10 mL of dichloromethane, 1 M sodium hydroxide, ethanol, pH 7 water (three times) using a neodymium magnet for 5 min each step to pelletize nanoparticles and remove the supernatant.</li>
		<li>Adjust the nanoparticle suspension in water to pH 2 using 1 M hydrochloric acid, and then centrifuge in 100 kDa ultracentrifugal filters at 2,200 x g for 10 min.</li>
		<li>Remove the filtrate, add 12 mL of 10-2 M hydrochloric acid to the nanoparticles solution and centrifuge in 100 kDa ultracentrifugal filters at 2,200 x g for 10 min (three times). Then dilute the nanoparticles solution within 10 mL of 10-2 M hydrochloric acid before coating.</li>
	</ol>
	</li>
	<li>Coating
	<ol>
		<li>Dilute 175 mg of citric acid and polyacrylic acid in water at pH 2 in a 100 mL glass vial and adjusted the pH to 2 with a 1 M hydrochloric acid solution.</li>
		<li>Add the 10 mL nanoparticles aqueous dispersion to the coating molecule solution, corresponding to a mass ratio of 5 between the coating molecule and the nanoparticles. Agitate for 2 h under magnetic stirring at room temperature.</li>
		<li>After the reaction, adjust the pH to 7 with 1 M sodium hydroxide.</li>
		<li>Centrifuge the nanoparticle solution 3 times with deionized&#160;(DI) water in 100 kDa ultracentrifugal filters at 2,200 x g for 10 min; remove the filtrate and dilute the nanoparticles solution in 12 mL of DI water.</li>
	</ol>
	</li>
</ol>
2. Culture and magnetic labeling of stem cells
<ol>
	<li>Culture human mesenchymal stem cells (MSC) in complete Mesenchymal Stem Cell Growth Medium (MSCGM) at 37 &#176;C and 5% CO2. When the cells are at 90% confluence, add 10 mL of trypsin-EDTA pre-warmed at 37&#176;C per 150 cm&#178; flask and leave for 2-3 minutes to detach the cells.
	<ol>
		<li>To know when the cells are detached, observe them with a bright-field microscope. Resuspend the detached cells in MSCGM and divide them in four 150 cm&#178; flasks. Amplify the cells this way until passage 4 to 5.</li>
	</ol>
	</li>
	<li>Prepare the solution of magnetic nanoparticles for cell labeling: disperse the chosen concentration of iron oxide nanoparticles in serum free Roswell Park Memorial Institute&#160;medium (RMPI-1640) without glutamine. RPMI is used for nanoparticle incubation (and the related rinsing steps) as it has a lower ionic strength than DMEM and better prevents nanoparticle aggregation events.</li>
	<li>When the cells are at passage 4 or 5 and 90% confluent, remove the MSCGM medium, rinse the cells with serum free RPMI (without glutamine) and add 10 mL of iron oxide nanoparticles solution per 150 cm2 culture flask, which is the minimum volume required to cover all the cells.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for 30 min and then discard the nanoparticle solution. Wash once with serum free RPMI-1640 (no glutamine) and incubate with Dulbecco&#8217;s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin (25 mL per 150 cm2 flask) overnight at 37 &#176;C and 5% CO2 to allow a complete internalization of the nanoparticles.</li>
</ol>
3. Formation of stem cell-spheroids
<ol>
	<li>Freshly prepare cell-spheroid culture medium composed of DMEM high glucose with L-glutamine supplemented with 50 &#181;M L-ascorbic acid 2-phosphate, 0.1 &#181;M dexamethasone, 1 mM sodium pyruvate, 0.35 mM L-proline, and 1% universal culture supplement containing Insulin, human Transferrin and Selenous acid (ITS-Premix).</li>
	<li>Detach the magnetic MSCs by adding 10 mL of 0.05% trypsin-EDTA pre-warmed at 37 &#176;C per 150 cm2 flask for 2-3 minutes. When the cells are detached, immediately inactivate the trypsin by adding 1/3 of the volume of DMEM supplemented with 10% FBS and 1% penicillin-streptomycin pre-warmed at 37 &#176;C.</li>
	<li>Centrifuge the dissociated cells at 260 x g for 5 min, aspirate the media and re-suspend the cells in a small volume (less than 1 mL per 150 cm&#178; flask) of cell-spheroid culture medium such as having 200,000 cells in about 50 &#181;L of medium. Count the cell number using a hemocytometer and adjust the volume if needed.</li>
	<li>Add 1 mL of freshly prepared cell-spheroid culture medium in a 15 mL sterile conical centrifuge tube and add the volume of resuspended solution corresponding to 200,000 cells.</li>
	<li>Centrifuge these magnetically labeled cells at 180 x g for 3 min such as forming a cell pellet at the bottom of the tube and keep the supernatant, which is the cell culture medium.</li>
	<li>Slightly open the tubes to allow air exchange and incubate at 37 &#176;C with 5% CO2 for up to 21 days, culture time along which the cells form a cohesive 3D structure that results in a fully formed spheroid. Change the medium twice a week.</li>
	<li>On given days, wash the spheroids twice with cacodylate buffer made of 0.2 M cacodylate diluted in demineralized water and fix them using 2% glutaraldehyde in 0.1 M cacodylate buffer diluted in distilled water for 30 min at room temperature. Store the fixed spheroids in PBS until used for VSM measurement or TEM imaging. This fixation step stops all biological processes and allows long-term conservation.</li>
</ol>
4. Quantification of magnetic nanoparticles in solution and in cellulo using a vibrating sample magnetometer (VSM)
<ol>
	<li>Place either a given volume of magnetic nanoparticle solution (maximum 10 &#181;L) or a single cell-spheroid into the sample holder specifically designed to fit in the VSM.</li>
	<li>Insert the sample in the VSM and scan for sample offset. Place the sample at the position corresponding to the magnetization maximum.</li>
	<li>Perform the first measurement at a low magnetic field (between -1500 Oe and +1500 Oe) with a 20 Oe/s rate.</li>
	<li>Perform a second measurement at a high magnetic field (between -30 000 Oe and +30&#160;000 Oe) with a 200 Oe/s rate.</li>
</ol>
5. Transmission Electron Microscopy (TEM) analysis
<ol>
	<li>For the nanoparticle solutions, deposit 10 &#956;L of the aqueous solution onto a carbon-coated copper grid and let it dry at room temperature.</li>
	<li>For the nanoparticles internalized in cells, contrast the fixed cell-spheroids with Oolong Tea Extract (OTE) at 0.5% diluted in 0.1 M cacodylate buffer, post fix with 1% osmium tetroxide containing 1.5% potassium cyanoferrate and then dehydrate in graded ethanol baths, included in Epon. Slice ultrasections of 70 nm and deposit them onto cooper grids.</li>
	<li>Take images using an electron microscope at 80 kV with a magnification from 1k for the observation of entire cells to 40k for the observation of endosomal compartments.</li>
</ol>

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The authors have nothing to disclose.

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&amp;deg;C</td></tr><tr><td>Dichloromethane &amp;ge;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 (&gt; 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&amp;deg;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&amp;micro;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&amp;deg;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...

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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 ...

using-magnetometry-to-monitor-cellular-incorporation-subsequent

Research

JoVE Journal

Bioengineering

4.5K Views.  Complexes MSC, UMR 7057, CNRS & University Paris Diderot. Deze video introduceert een snelle en efficiënte methode voor de synthese van magnetische nanodeeltjes en het gebruik van magnetometrie om transformatie in de biologische omgeving te fotograferen. Magnetische nanodeeltjes hebben meer aandacht getrokken voor biomedische toepassingen, en het is belangrijk om te begrijpen dat het lot ooit geïnternaliseerd was in cellen. Dit wordt hier bereikt door het cellulaire magnetisme te meten.Voor zowel magnetische nanodeeltjessynthese als metinge...

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

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

Medicine

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

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

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

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

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

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

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

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

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.

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 ...

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 ...

Chemistry

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

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

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

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

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 ...

Fluorescent Assay for Detection of Novel Iron Chelators

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

Author Spotlight: Assessing the Impact of Novel Iron Chelators on Cancer Cell Metabolism

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

Cancer Research

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

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

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