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&#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 ...

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

Research

JoVE Journal

Bioengineering

4.1K Views.  Complexes MSC, UMR 7057, CNRS & University Paris Diderot. 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).

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

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 infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with 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 antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

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

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

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

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

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

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

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

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

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

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

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

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 ...

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 widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

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 various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

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

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

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

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

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

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

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...