Name: catalytic scavenging of plant reactive oxygen species in vivo by anionic cerium oxide nanoparticles
Uploaded: 2018-08-26T15:00:00
Description: Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

This work was supported by the University of California, Riverside and USDA National Institute of Food and Agriculture, Hatch project 1009710 to J.P.G. This material is based upon work supported by the National Science Foundation under Grant No. 1817363 to J.P.G.

1. Growing A. thaliana Plants
<ol>
	<li>Sow A. thaliana seeds in 5 cm x 5 cm disposable pots filled with standard soil mix. Put 32 of these pots into a plastic tray filled with water (~ 0.5 cm depth) and transfer the plastic tray with the plants into a plant growth chamber.
	<ol>
		<li>Set the growth chamber settings as follows: 200 &#181;mol/ms photosynthetic active radiation (PAR), 24 &#177; 1 &#176;C day and 21 &#177; 1 &#176;C night, 60% humidity, and 14/10 h day/night light regime, respectively.<...

<ol>
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In this protocol, we describe PNC synthesis, characterization, fluorescent dye labeling, and confocal imaging of the nanoparticles within plant mesophyll cells to exhibit their in vivo ROS scavenging activity. PNC are synthesized from a mixture of cerium nitrate and PAA solution in ammonium hydroxide. PNC are characterized by absorption spectrophotomery and the concentration determined using Beer-Lamberts law. Zeta potential measurements confirmed the negatively charged surface of PNC for enhancing delivery to c...

Cerium oxide nanoparticles (nanoceria) are widely used in living organisms, from basic research to bioengineering, due to their distinct catalytic reactive oxygen species (ROS) scavenging ability1,2,3. Nanoceria have ROS scavenging abilities due to a large number of surface oxygen vacancies that alternate between two oxidation states (Ce3+ and Ce4+) 4,5,6. The Ce3+ dangling bonds effectively scavenge ROS while the lattice strains at the nanoscale promote the regeneration of these defect sites via redox cycling reactions7. Nanoceria have also been recently used for studying and engineering plant function8,9. Plants under abiotic stress experience accumulation of ROS, causing oxidative damage to lipids, proteins, and DNA10. In A. thaliana plants,&#160;nanoceria catalytic scavenging of ROS in vivo leads to improved plant photosynthesis under high light, heat, and chilling stresses8. Applying nanoceria to soil also increases shoot biomass and grain yield of wheat (Triticum aestivum)11; canola (Brassica napus) plants treated with nanoceria have higher plant biomass under salt stress12.
Nanoceria offer&#160;bioengineers and plant biologists a nanotechnology-based tool to understand abiotic stress responses and enhance plant abiotic stress tolerance. Nanoceria&#39;s&#160;in vivo ROS&#160;scavenging capabilities are independent of plant species, and the facile delivery into plant tissues has the potential to enable broad application outside of model organisms. Unlike other genetically-based methods, nanoceria do&#160;not require generating plant lines with the overexpression of antioxidant enzymes for higher ROS scavenging ability13. Leaf lamina infiltration of nanoceria to plants is a&#160;practical approach&#160;for lab-based research.
The overall goal of this protocol is to describe 1) the synthesis and characterization of negatively charged poly (acrylic) acid nanoceria (PNC), 2) the delivery and tracking of PNC throughout leaf cells, and 3) the monitoring of PNC-enabled ROS scavenging in vivo. In this protocol, negatively charged poly (acrylic) acid&#160;nanoceria (PNC) are synthesized and characterized by their absorption spectrum, hydrodynamic diameter, and zeta potential. We describe a simple leaf lamina infiltration method to deliver PNC into plant leaf tissues. For in vivo imaging of nanoparticle distribution within mesophyll cells, a fluorescent dye (DiI) was used to label PNC (DiI-PNC) and observe the nanoparticles via confocal fluorescence microscopy. Finally, we explain how to monitor in vivo PNC ROS scavenging through confocal microscopy.

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			<td height="21" style="height:21px;width:567px;">Cerium (III) nitrate hexahydrate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">238538-100G</td>
			<td style="width:265px;"></td>
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			<td height="21" style="height:21px;width:567px;">Molecular Biology Grade Water, Corning</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">45001-044&#160;</td>
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			<td height="21" style="height:21px;width:567px;">Falcon 50 mL Conical Centrifuge Tubes</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">14-959-49A</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">Poly (acrylic acid) 1,800 Mw</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">323667-100G</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">Fisherbrand Digital Vortex Mixer</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">02-215-370</td>
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			<td height="21" style="height:21px;width:567px;">Fisherbrand Digital Vortex Mixer Accessory, Insert Retainer</td>
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			<td style="width:165px;">02-215-395</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">Ammonium hydroxide solution</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">05002-1L</td>
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			<td style="width:165px;">13912-149&#160;</td>
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			<td height="21" style="height:21px;width:567px;">RCT basic</td>
			<td style="width:228px;">IKA</td>
			<td style="width:165px;">3810001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Eppendorf Microcentrifuge 5424</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">80094-126</td>
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			<td height="21" style="height:21px;width:567px;">Amicon Ultra-15 Centrifugal Filter Units</td>
			<td style="width:228px;">Millipore-Sigma</td>
			<td style="width:165px;">UFC901024</td>
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			<td height="21" style="height:21px;width:567px;">Allegra X-30 Series Benchtop Centrifuge</td>
			<td style="width:228px;">Beckman Coulter</td>
			<td style="width:165px;">B06314</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">UV-2600 Sptecrophotometer</td>
			<td style="width:228px;">Shimadzu</td>
			<td style="width:165px;">UV-2600 120V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Whatman Anotop 10 syringe filter</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">WHA68091102</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">BD Disposable Syringes with Luer-Lok Tips</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">14-829-45</td>
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			<td height="21" style="height:21px;width:567px;">Zetasizer Nano S</td>
			<td style="width:228px;">Malvern Panalytical</td>
			<td style="width:165px;">Zen 1600</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">1,1&#8242;-Dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine perchlorate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">42364-100MG</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Dimethyl Sulfoxide, ACS</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">BDH1115-1LP</td>
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			<td height="21" style="height:21px;width:567px;">Sunshine Mix #1 LC1</td>
			<td style="width:228px;">Green Island Distributors, Inc</td>
			<td style="width:165px;">5212601.CFL080P</td>
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			<td height="21" style="height:21px;width:567px;">Adaptis 1000</td>
			<td style="width:228px;">Conviron</td>
			<td style="width:165px;">A1000</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">TES, &#62;99% (titration</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">T1375-100G</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">Magnesium chloride</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">M8266-1KG</td>
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			<td height="21" style="height:21px;width:567px;">Air-Tite All-Plastic Norm-Ject Syringe</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">14-817-25</td>
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			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">06-666A</td>
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			<td height="21" style="height:21px;width:567px;">Carolina Observation Gel</td>
			<td style="width:228px;">Carolina</td>
			<td style="width:165px;">132700</td>
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			<td height="21" style="height:21px;width:567px;">Corning microscope slides, frosted one side, one end</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">CLS294875X25-72EA</td>
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			<td height="21" style="height:21px;width:567px;">Cork Borer Sets with Handles</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">S50166A</td>
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			<td height="21" style="height:21px;width:567px;">Perfluorodecalin</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">P9900-25G</td>
			<td></td>
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			<td height="21" style="height:21px;width:567px;">Micro Cover Glasses, Square, No. 1</td>
			<td style="width:228px;">VWR</td>
			<td style="width:165px;">48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Leica Laser Scanning Confocal Microscope TCS SP5</td>
			<td style="width:228px;">Leica Microsystems</td>
			<td style="width:165px;">TCS SP5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">2&#8242;,7&#8242;-Dichlorofluorescin diacetate</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">D6883-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Dihydroethidium</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">D7008-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Fisherbrand Premium Microcentrifuge Tubes: 1.5 mL</td>
			<td style="width:228px;">Fisher Scientific</td>
			<td style="width:165px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Eppendorf Uvette cuvettes</td>
			<td style="width:228px;">Sigma-Aldrich</td>
			<td style="width:165px;">Z605050-80EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:567px;">Chlorophyll meter&#160;</td>
			<td style="width:228px;">Konica Minolta</td>
			<td style="width:165px;">SPAD-502</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

PNC synthesis and characterization. 
PNC were synthesized, purified and characterized following the method described in Protocol Section 2. Figure 1A shows the coloration of the solutions of cerium nitrate, PAA, the mixture of cerium nitrate and PAA, and PNC. A color change from white to light yellow is seen after PNC is synthesized. After purification with a 10 kDa filter, PNC were characterize...

Watch this Scientific Journal Video about Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles at JoVE.com

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.

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 ...

The ability of cerium oxide nanoparticles to catalytically scavenge reactive oxygen species in vivo provides a unique tool to understand and engineer mechanisms of plant signaling and tolerance to stress. Reactive oxygen species play a key role in plants by acting as signal molecules at low levels and damaging molecules at high levels. The main advantage of this technique is the potential to be applied to diverse wild type plant species with broad leaves and leaf structure, like Arabidopsis thaliana.After growing the Arabidopsis thaliana plants, weigh out 1.08 grams of Cerium nitrate and dissolve it in 2.5 milliliters of biology-grade water in a 50-milliliter conical tube. Weigh out 4.5 grams of Poly acid and dissolve it in five milliliters of biology-grade water in a different 50-milliliter conical tube. Combine these two solutions and then use a digital vortex mixer to mix them thoroughly at 2, 000 rpm for 15 minutes.In a fume hood, transfer 15 milliliters of a 7.2-molar ammonium hydroxide solution to a 50-milliliter glass beaker. Begin stirring at 500 rpm and at room temperature. Add the Cerium nitrate and Poly acid mixture drop wise.Cover the beaker with a piece of paper to prevent substantial loss of the solution and let it stir for 24 hours. After this, transfer the resulting solution to a 50-milliliter conical tube. Centrifuge at 3, 900 times g for one hour to remove any debris or large agglomerates.Then, transfer the supernatant into three 50-milliliter, 10-kilodalton filters. Fill the remainder of each filter with molecular-grade water, resulting in a total dilution of 45 milliliters. Centrifuge at 3, 900 times g for 15 minutes.Repeat this process at least six times. Using a UV-vis spectrophotometer, measure the absorbance of each eluent from 220 nanometers to 700 nanometers to ensure that no free polymers or other reagents are present in the final PNC solution. Use a five-milliliter syringe to take up the collected PNC solution.Next, filter the solution against a 20-nanometer pore-sized syringe filter and collect the filtered solution in a 50-milliliter conical tube. Transfer a diluted final PNC solution into a plastic cuvette and measure its absorbance from 220 nanometers to 700 nanometers. Using a particle sizer, measure the hydrodynamic diameter of the synthesized PNC, then store the final PNC solution in a refrigerator at four degrees Celsius until ready to use.After labeling the PNC with DiI fluorescent dye, transfer 0.9 milliliters of a solution containing 0.5 millimolar of either the PNC solution or the DiI-PNC solution to a tube. Add 0.1 milliliter of infiltration buffer and mix by vortexing. Prepare a 10-millimolar TES infiltration buffer as a negative control.Next, transfer 0.2 milliliters of the sample and buffer mixture to a one-milliliter sterile needleless syringe. Tap to remove any possible air bubbles. Just before infiltration, retrieve the plant from the growth chamber.Using a chlorophyll meter, measure the chlorophyll content in the A.thaliana leaves. Measure each leaf with three replicates as outlined in the text protocol. Choose leaves with similar chlorophyll content.Gently press the tip of the needleless syringe against the bottom of the leaf lamina and depress the plunger to infiltrate the leaves. Using a delicate task wiper, gently wipe off any excess solution that remains on the surface of the leaf lamina, then label the plant. Keep the infiltrated plants on the bench to incubate for three hours.First, place a pea-sized amount of observation gel onto a glass slide, roll the gel and spread it out until it is approximately two centimeters in diameter and one millimeter thick. Using a cork borer with a diameter of 0.3 centimeters, cut out a circular section in the center of the gel to create a well. Fill the well completely with PFD, which will provide a deeper and better confocal imaging resolution.Then, use a cork borer with a diameter of 0.2 centimeters to collect leaf disks from the previously-infiltrated A.thaliana plants. Mount each leaf disk in a PFD-filled well with the infiltrated side facing up. After this, place a square cover slip on top of the leaf disk.Press gently and evenly on the cover slip to seal with the well of observation gel and ensure that no air bubbles remain trapped. To begin, prepare 25 micromolar of H2DCFDA dye and 10 micromolar of DHE dye in TES infiltration buffer in separate 1.5-milliliter microcentrifuge tubes. Using a cork borer with a diameter of 0.2 centimeters, collect leaf disks from the adapted PNC-infiltrated A.thaliana plants.Use the sharp tip of forceps to make three to four holes on each leaf disk to accelerate the dye-loading process. Next, load an equal amount of leaf disks into each dye tube. Incubate in darkness for 30 minutes.After this, rinse the leaf disks with double-distilled water three times. Use observation gel to mount each leaf to a glass slide as previously described. Load the slide on a confocal microscope and manually focus to a region of leaf mesophyl cells as outlined in the text protocol, then expose the leaf disks to a UVA laser at 405 nanometers for three minutes to generate ROS.Record the ROS signal intensity change in time series per leaf disk. Image the leaf disk using a 40X water objective and the confocal microscope settings shown here. Use a plant infiltrated with only infiltration buffer solution as the negative control.After purification, the synthesized PNC is characterized using a UV-vis spectrophotometer and is seen to have a peak of absorbance at 271 nanometers. The purification process is confirmed by measuring the final eluent. PNC samples are then labeled with a fluorescent DiI dye to determine the distribution of the nanoparticles in vivo.Three peaks are observed for the DiI-labeled PNC, while a measurement of the final eluent confirms that the non-reacted chemicals are washed out during purification. The distribution of DiI-PNC in leaf mesophyl cells is determined via confocal imaging. While no DiI dye signals are detected in the control samples the DiI-PNC is observed co-localized with chloroplasts in the infiltrated leaves.In vivo ROS scavenging enabled by PNC is monitored in the leaf disk by measuring the fluorescence intensity changes in the DHE and DCF dyes. After three minutes of UV stress, the PNC-infiltrated leaves show significantly less intensity for both dye types when compared to the no-nanoparticle buffer control. A CAT mimetic activity assay reveals a decrease in the resorufin level in the mixture containing PNC.This level is indicative of the hydrogen peroxide level, which therefore is also decreasing, and confirms the CAT mimetic activity of the synthesized PNC. This protocol for synthesizing nanoceria is a simple, stepwise procedure that generates cerium oxide nanoparticles with controlled size, charge, and ROS scavenging abilities. Plant genetic modification methods for ROS manipulation in vivo are currently limited to plant model species.In contrast, nanoceria ROS scavenging has the potential to be applied to diverse wild type plant species both in the laboratory and in the field.

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Research

JoVE Journal

Bioengineering

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

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

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

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

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

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

Novel ROS Quantification Using Dihydroethidium in Liver Cancer Cells

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

Author Spotlight: High-Throughput Measurement of Intracellular ROS Levels in Hepatocellular Lines

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...