Name: fabrication of a functionalized magnetic bacterial nanocellulose with iron oxide nanoparticles
Uploaded: 2016-05-26T13:00:00
Description: Watch this Scientific Journal Video about Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles at JoVE.com

This work was funded by Department of Defense under contract No. W81XWH-11-2-0067

1. Preparation of Bacterial Nanocellulose (BNC)
Note: All of the steps are performed under aseptic conditions, unless otherwise indicated.
<ol>
	<li>Prepare culture medium.
	<ol>
		<li>Prepare 500 ml of liquid culture medium by combining 25 g of yeast extract, 15 g of peptone, 125.0 g of mannitol, and 500 ml of high purity water. Autoclave this mixture at 120 &#176;C for 20 min and store at 4 &#176;C.</li>
		<li>Prepare 100 ml of semisolid media by adding 15 g of agar to 5.0 g of yeast extra...

<ol>
	<li>Saxena, I. M., Brown, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosynthesis+of+bacterial+cellulose.">Biosynthesis of bacterial cellulose.</a> Bacterial Nanocellulose: A Sophisticated Multifunctional Material. , 1-18 (2012).</li><li>Chan-Park, M. B., Shen, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+control+of+vascular+smooth+muscle+cell+morphology+and+phenotype+for+functional+tissue-engineered+small-diameter+blood+vessels.">Biomimetic control of vascular smooth muscle cell morphology and phenotype for functional tissue-engineered small-diameter blood vessels.</a> J.Biomed.Mater.Res.A. 88, 1104-1121 (2009).</li><li>Barud, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocellulose-based+flexible+magnetic+paper.">Biocellulose-based flexible magnetic paper.</a> J. Appl. Phys. 117, (2015).</li><li>M&#228;rtson, M., Viljanto, J., Hurme, T., Laippala, P., Saukko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+cellulose+sponge+degradable+or+stable+as+implantation+material?+An+in+vivo+subcutaneous+study+in+the+rat.">Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat.</a> Biomaterials. 20, 1989 (1999).</li><li>Ill&#233;sa, E., Tomb&#225;cza, E., Szekeresa, M., T&#243;tha, I., Szab&#243;b, &. #. 1. 9. 3. ;., Iv&#225;n, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+carboxylated+PEG-coating+on+magnetite+nanoparticles+designed+for+biomedical+applications.">Novel carboxylated PEG-coating on magnetite nanoparticles designed for biomedical applications.</a> J. Magn. Magn. Mater. 380, 132 (2015).</li><li>Torrisi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preventing+corona+effects:+multiphosphonic+acid+poly(ethylene+glycol)+copolymers+for+stable+stealth+iron+oxide+nanoparticles.">Preventing corona effects: multiphosphonic acid poly(ethylene glycol) copolymers for stable stealth iron oxide nanoparticles.</a> Biomacromolecules. 15, 3171 (2014).</li><li>Cai, Z., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cellulose/poly(ethylene+glycol)+composite:+characterization+and+first+evaluation+of+biocompatibility.">Bacterial cellulose/poly(ethylene glycol) composite: characterization and first evaluation of biocompatibility.</a> Cellulose. 17, 83 (2010).</li><li>Wu, W., He, Q., Jiang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+iron+oxide+nanoparticles:+synthesis+and+surface+functionalization+strategies.">Magnetic iron oxide nanoparticles: synthesis and surface functionalization strategies.</a> Nanoscale Res. Lett. 3, 397-415 (2009).</li><li>Ulbricht, J., Jordan, R., Luxenhofer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+biodegradability+of+polyethylene+glycol,+polypeptoids+and+poly+(2-oxazoline)s.">On the biodegradability of polyethylene glycol, polypeptoids and poly (2-oxazoline)s.</a> Biomaterials. 35, 4848 (2014).</li><li>Azqueta, A., Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+comet+assay:+a+comprehensive+guide+to+measuring+DNA+damage+and+repair.">The essential comet assay: a comprehensive guide to measuring DNA damage and repair.</a> Arch. Toxicol. 87 (6), 949-968 (2013).</li><li>Scherner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+application+of+tissue-engineered+blood+vessels+of+bacterial+cellulose+as+small+arterial+substitutes:+proof+of+concept.">In vivo application of tissue-engineered blood vessels of bacterial cellulose as small arterial substitutes: proof of concept.</a> J. Surg. Res. 189, 340 (2014).</li><li>Bodin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cultivation+conditions+on+mechanical+and+morphological+properties+of+bacterial+cellulose+tubes.">Influence of cultivation conditions on mechanical and morphological properties of bacterial cellulose tubes.</a> Biotechnol Bioeng. 97, 425 (2007).</li><li>Zaborowska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microporous+bacterial+cellulose+as+a+potential+scaffold+for+bone+regeneration.">Microporous bacterial cellulose as a potential scaffold for bone regeneration.</a> Acta Biomaterialia. 6, 2540 (2010).</li><li>Karimi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+the+mechanical+properties+of+the+umbilical+vein+and+umbilical+artery+under+uniaxial+loading.">A comparative study on the mechanical properties of the umbilical vein and umbilical artery under uniaxial loading.</a> Artery Res. 8, 51 (2014).</li><li>Lina, F., Ping, Z., Shengmin, Z., Guang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+bacterial+nanocellulose-based+uniform+wound+dressing+for+large+area+skin+transplantation.">Evaluation of bacterial nanocellulose-based uniform wound dressing for large area skin transplantation.</a> Mater. Sci. Eng. C. 33, 2995 (2013).</li><li>Olsson, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+flexible+magnetic+aerogels+and+stiff+magnetic+nanopaper+using+cellulose+nanofibrils+as+templates.">Making flexible magnetic aerogels and stiff magnetic nanopaper using cellulose nanofibrils as templates.</a> Nature Nanotech. 5 (8), 584-588 (2010).</li><li>Torre, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+force+microscopy+and+energy+loss+imaging+of+superparamagnetic+iron+oxide+nanoparticles.">Magnetic force microscopy and energy loss imaging of superparamagnetic iron oxide nanoparticles.</a> Sci. Rep. 1 (202), 1-8 (2011).</li></ol>

The thickness and size of the BNC pellicle can be easily manipulated by changing the incubation time and the size of the flask in which it is grown during static cultivation. The microproperties of the BNC, such as porosity, can be modified by changing the oxygen ratio in the static culture. Higher oxygen concentrations yield tougher BNC11. A. Bodin and coworkers produced tubes of BNC with a burst pressure up to 880 mm Hg by changing the oxygen ratio from atmospheric oxygen to 100% oxygen during the fermentati...

The bacterian nanocellulose (BNC) is synthesized by Acetobacter xylinum strain, also known as Gluconacetobacter xylinus, and deposited in the form of films or pellicles on the air-liquid interface during stationary culture. These BNC pellicles adopt the form of the container where they are grown, and their thickness depends on the number of days in culture. A. xylinus uses the glucose in the medium for the synthesis of the cellulose microfibrils through a process of polymerization and subsequent crystallization. The polymerization of the glucose residues is carried out at the bacterial extracellular membrane where glucan chains are extruded from single pores distributed over the cell envelope. The crystallization of the cellulose microfibrils occurs in the extracellular space with the formation of glucan chain sheets by van der Waals bonding followed by stacking of the sheets by H-bonding1.
Magnetic nanoparticles integrated to a BNC matrix can be manipulated easily by an external magnetic field in order to increase the force necessary to direct and confine smooth muscle cells (SMCs) containing magnetic nanoparticles, at the damaged site of the arterial wall. This strategy keeps the SMCs away from other tissues, and holds the cells in place against the force exerted by the blood flow. It has been shown that SMCs play an important role in the vasoelasticity of the blood vessel, where they form abundant layers located mainly in the tunica media2.
The method used for the synthesis of MBNC involves BNC pellicle immersed and stirred in a solution of iron(III) chloride hexahydrate and iron(II) chloride tetrahydrate at 80 &#176;C. Ammonium hydroxide is added to form iron oxide nanoparticles inside the BNC mesh. The addition of ammonium hydroxide changes the color of the solution from orange to black. The IONPs compact together along the BNC fibrils with a non-uniform distribution.
This protocol focuses on the design of a bacterial nanocellulose-magnetic nanoparticle pellicle, which we have named magnetic bacterial nanocellulose (MBNC), which is intended to use as a substitute for missing, damaged or injured small-diameter blood vessels. H. S. Barud and coworkers have recently published a similar work to produce a BNC-based flexible magnetic paper by mixing BNC pellicles in a stable aqueous dispersion of PEG and superparamagnetic iron oxide nanoparticles3. Here, we describe the production of bacterial cellulose and its impregnation in situ with magnetic nanoparticles. A cytotoxicity assay based on detection of single DNA strand breaks was used to test the biocompatibility of the BNC and MBNC pellicles.

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Glucoacetobacter Xylinus</td>
			<td style="width:167px;">ATCC</td>
			<td style="width:101px;">700178</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Agar</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:101px;">A1296-500G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">D-Mannitol Bioxtra</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">M9546-250G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Yeast Extract</td>
			<td style="width:167px;">BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bacteriological Peptone</td>
			<td>Sigma Aldrich</td>
			<td>P0556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Sodium Hydroxide, 50% Solution In Water</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">158127-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Iron(III) Chloride Hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">236489-100G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Ammonium Hydroxide&#160;</td>
			<td>Macron Fine Chemicals</td>
			<td style="width:101px;">6665-46</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Poly(Ethylene Glycol), Average Mn 400</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">202398-250G&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Iron (II) chloride tetrahydrate</td>
			<td>Sigma Aldrich</td>
			<td style="width:101px;">44939-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Disposable petri dish</td>
			<td>Sigma Aldrich</td>
			<td>BR452000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Disposable Inoculating Loop</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td>22-363-604&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Anhydrous Calcium Sulfate</td>
			<td style="width:167px;">W.A. Hammond Drierite&#160;</td>
			<td style="width:101px;">13001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">High vacuum grease</td>
			<td>Sigma Aldrich</td>
			<td>Z273554-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Laboratory pipetting needle with 90&#176; blunt ends</td>
			<td>Sigma Aldrich</td>
			<td>CAD7937-12EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">pH test strips&#160;&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P4786-100EA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Round-bottom three neck angle type distilling flask</td>
			<td>Sigma-Aldrich</td>
			<td>CLS4965250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Silicone oil for oil baths</td>
			<td>Sigma-Aldrich</td>
			<td style="width:101px;">85409-250ML&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Drying Tube</td>
			<td style="width:167px;">Chemglass</td>
			<td>CG-1295-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Septum Stopper, Sleeve Type</td>
			<td style="width:167px;">Chemglass</td>
			<td>CG-3022-98</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Magnetic stir bar</td>
			<td style="width:167px;">Chemglass</td>
			<td>CG-2001-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Condenser</td>
			<td style="width:167px;">Chemglass</td>
			<td>CG-1218-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Temperature Controller</td>
			<td style="width:167px;">BriskHeat</td>
			<td>SDC120JC-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Stirring Hotplate</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td>11-100-49SH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Comet Assay Kit</td>
			<td style="width:167px;">Trevigen</td>
			<td>4250-050-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">SYBR Gold Nucleic Acid Gel Stain</td>
			<td style="width:167px;">Life Technologies</td>
			<td>S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">bio-AFM</td>
			<td style="width:167px;">JPK Instruments</td>
			<td colspan="2">NanoWizard 4a BioScience AFM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Nanoindenter</td>
			<td style="width:167px;">Micro Materials Ltd</td>
			<td colspan="2">Multi-module mechanical tester&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Scanning electron microscopy (SEM)</td>
			<td>Hitachi High Technologies America</td>
			<td>Hitachi S-4800</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The incubation period of G. xylinus was a total of 9 days, but the pellicles began to form earlier and were evident after about 2 days. The macroscopic appearance of the BNC is displayed in Figure 1, the shape of which mimics that of the dish-grown culture. Figure 2 describes the process for producing BNC-IONP pellicles, which summaries the main steps involved in the protocol above as well as the configuration of the main compone...

Watch this Scientific Journal Video about Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles at JoVE.com

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

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 ...

The overall goal of this procedure is to synthesize bacterial nano-cellulose, or BNC, and render it magnetic by impregnating in situ with iron oxide nano-particles. Magnetic BNC membranes are designed to locally attact and retain magnetized cells against hemodynamic flow. The main advantage of the magnetic BNC synthesized in this procedure is that it provides a minimal invasive treatment near brain aneurysms.Convention treatments of brain aneurysms such as clipping, and neural and vascular core emulisation are invasive and traumatic and not suitable in most patients with increased risks. The implications of this technique extend towards therapy of vascular diseases, because vascular grafts, made from magnetic scaffolds can speed healing by improving cell retention at the site of injury. Begin this procedure with preparation of the liquid culture medium as described in the text protocol.Transfer two milliliters of the liquid culture medium into each well of a 24 well tissue culture plate. Take two colonies with an inoculating needle from the inoculated petri dishes in step, and place them into the first well of the tissue culture plate. Repeat the same procedure for the remaining 23 wells.Incubate the tissue culture plate at 30 degrees Celsius for seven days. This will yield a total of 24 BNC pellicles with a diameter of 16 mm and a thickness of approximately 2 to 3 mm. Collect the BNC pellicles from the growth media and sterilize them in 200 milliliters of one percent sodium hydroxide solution for one hour at 50 degrees Celsius to remove all traces of G.Xylinus.Discard the solution, and add 200 milliliters of freshly prepared one percent sodium hydroxide solution. Repeat the same process once more or until the BNC pellicles in solution acquire a translucent appearance. Rinse the BNC pellicles with water three times, and store them in high purity water at room temperature.Make sure the BNC pellicles are completely submerged in the water and are not allowed to dry at any time. Then, autoclave the BNC pellicles at 121 degrees Celsius for 20 minutes. To begin synthesis, bubble 1000 milliliters of high purity water with nitrogen gas to remove any dissolved oxygen in the water and replace it with nitrogen.Use a three neck round-bottom flask to prepare a solution in a 2 to 1 molar ratio of iron 3 chloride hexahydrate and iron 2 chloride tetra-hydrate, diluted with the de-oxygenated high purity water. Use 2 necks of the vessel to provide a constant entrance and output of nitrogen gas by connecting the nitrogen gas supply to a needle punched in a septum stopper and fixed to the vessel's neck. Place one BNC pellicle in the vessel with the reactants.Make sure the sample is completely submerged in the liquid. Then, fill all of the glass joints with vacuum grease. Connect the remaining neck of the vessel to a condenser tube topped with a drying tube filled with anhydrous calcium sulphate.Run water through the condenser tube. Next, heat the solution in a silicone oil bath to 80 degrees Celsius, using a stirring hot plate and hold this temperature. Use a small magnetic stir bar to mix the reactants at 350 R.P.M.for 5 minutes.Make sure the BNC is appropriately impregnated with the ferrous solution and the reactants are completely dissolved. Increase the stirring velocity to 700 R.P.M.In a time interval of five minutes, add five milliliters of ammonium hydroxide to the 10 milliliters of ferrous solution using a pipetting needle that has been punched in a septum stopper. After addition of the ammonium hydroxide, the color of the solution changes from yellow-orange to black.Continue stirring the solution at 80 degrees Celsius for another five minutes. Avoid high speed stirrings to maintain the integrity of the sample. Lower the temperature of the solution to 30 degrees Celsius using the temperature control bottom of the stirring hot plate and keep stirring for another five minutes.Then, turn off the hot plate. At this point, the iron oxide nanoparticles have been incorporated into the BNC mesh. Next, cool the mixture down to room temperature and transfer it to a vessel flask to separate the magnetic nano-particles, or MNPs, and BNC with a strong permanent magnet.To do this, keep the magnet close to the vessel to hold the MNPs and BNC in place while decanting the supernatant. Re-suspend the MNPs and BNC in 100 milliliters of water. Gently shake the solution to remove all the MNPs that are not strongly incorporated into the BNC.Then, decant the supernatant again by holding the MNPs and the BNC in place using the magnet. Wash the MNPs and the BNC several times with water until the supernatant reaches neutral PH, as measured using a colorimetric strip. Separate the magnetic functionalized BNC, or MBNC, from the MNPs, using tweezers, and rinse the MBNCs several times with water until the water runs clear.Sterilize the MBNC by exposing it overnight to U.V.Store the MBNC in 20 milliliters of deoxygenated high purity water that has been autoclaved at 120 degrees Celsius for 20 minutes. Aseptically, immerse the sample in one percent of polyethylene glycol, or PEG, and stir for two hours at room temperature. Coating with PEG improves the biocompatibility and stability of the iron oxide nano-particles deposited in the BNC as it is distributed over the MBNC three-D network.The macroscopic appearance of the BNC is shown here. The culture vessel gives the shape of the pellicles. Scanning electron microscopy reveals that fine ribbons form the micro-structure of the BNC with approximately 50 nanometers in diameter, which create open pores across the entire network.Magnetic functionalization of the BNC gives a less compacted fibril structure as compared to the BNC. The iron oxide nano-particles are preferentially located between the pores formed by fibril interlacing. Atomic force microscopy topography detected the magnetic force gradient characterized by two domains across the MBNC surface.A high intensity magnetic field, shown in yellow, and a weak intensity magnetic field, shown in green. The presence of magnetic domains observed with MFM is confirmed by vibrating sample magnetometer data which shows that the iron oxide nano-particles are superparamagnetic, hence rendering the BNC magnetic. The integrity of the DNA on human aortic smooth muscle cells is preserved after a 24 hour incubation period, when cultured in the absence, and presence, of BNC or MBNC, as evident when compared to cells that underwent hydrogen peroxide treatment.After watching this video, you should have a good understanding of how to render a bacterial nanocellulose magnetic for applications in damaged blood vessel reconstruction. We first had the idea for this method when we needed to induce a density gradient of magnetic nano-particles that had to be dispersed within the complex matrix of the bacterial nanocellulose network. Integrating the iron oxide nano-particles in this network required that we conducted a method during synthesis of the BNC.Generally, individuals new to this method will struggle because there are a number of complex steps to impregnate iron oxide nano-particles into BNC.

fabrication-functionalized-magnetic-bacterial-nanocellulose-with-iron

Research

JoVE Journal

Bioengineering

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

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 ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era 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 ...

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 ...

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. Many studies have aimed at directing neurons using chemical and topographical cues. However, reports of organizational control on a micron-scale over large areas are scarce. Here, an effective method has been described for placing neurons in preset sites and guiding neuronal outgrowth with micron-scale resolution, using magnetic platforms embedded with micro-patterned, magnetic elements. It has been demonstrated that loading neurons with magnetic nanoparticles (MNPs) converts them into sensitive magnetic units that can be influenced by magnetic gradients. Following this approach, a unique magnetic platform has been fabricated on which PC12 cells, a common neuron-like model, were plated and loaded with superparamagnetic nanoparticles. Thin films of ferromagnetic (FM) multilayers with stable perpendicular magnetization were deposited to provide effective attraction forces toward the magnetic patterns. These MNP-loaded PC12 cells, plated and differentiated atop the magnetic platforms, were preferentially attached to the magnetic patterns, and the neurite outgrowth was well aligned with the pattern shape, forming oriented networks. Quantitative characterization methods of the magnetic properties, cellular MNP uptake, cell viability, and statistical analysis of the results are presented. This approach enables the control of neural network formation and improves neuron-to-electrode interface through the manipulation of magnetic forces, which can be an effective tool for in vitro studies of networks and may offer novel therapeutic biointerfacing directions.

This work presents a bottom-up approach to the engineering of local magnetic forces for control of neuronal organization. Neuron-like cells loaded with magnetic nanoparticles (MNPs) are plated atop and controlled by a micro-patterned platform with perpendicular magnetization. Also described are magnetic characterization, MNP cellular uptake, cell viability, and statistical analysis.

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. ...

Fabrication of a Crystalline Nanocellulose Embedded Agarose Biomaterial Ink for Bone Marrow-Derived Mast Cell Culture

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto which cells are deposited. Because many biomaterial inks can be potentially cytotoxic to primary cells, it is necessary to determine the biocompatibility of these hydrogel composites prior to their utilization in costly 3D tissue engineering processes. Some 3D culture methods, including bioprinting, require that cells be embedded into a 3D matrix, making it difficult to extract and analyze the cells for changes in viability and biomarker expression without eliciting mechanical damage. This protocol describes as proof of concept, a method to assess the biocompatibility of a crystalline nanocellulose (CNC) embedded agarose composite, fabricated into a 24-well culture system, with mouse bone marrow-derived mast cells (BMMCs) using flow cytometric assays for cell viability and biomarker expression.
After 18 h of exposure to the CNC/agarose/D-mannitol matrix, BMMC viability was unaltered as measured by propidium iodide (PI) permeability. However, BMMCs cultured on the CNC/agarose/D-mannitol substrate appeared to slightly increase their expression of the high-affinity IgE receptor (Fc&#949;RI) and the stem cell factor receptor (Kit; CD117), although this does not appear to be dependent on the amount of CNC in the bioink composite. The viability of BMMCs was also assessed following a time course exposure to hydrogel scaffolds that were fabricated from a commercial biomaterial ink composed of fibrillar nanocellulose (FNC) and sodium alginate using a 3D extrusion bioprinter. Over a period of 6-48 h, the FNC/alginate substrates did not adversely affect the viability of the BMMCs as determined by flow cytometry and microtiter assays (XTT and lactate dehydrogenase). This protocol describes an efficient method to rapidly screen the biochemical compatibility of candidate biomaterial inks for their utility as 3D scaffolds for post-print seeding with mast cells.

This protocol highlights a method to rapidly assess the biocompatibility of a crystalline nanocellulose (CNC)/agarose composite hydrogel biomaterial ink with mouse bone marrow-derived mast cells in terms of cell viability and phenotypic expression of the cell surface receptors, Kit (CD117) and high-affinity IgE receptor (Fc&#949;RI).

Three-dimensional (3D) bioprinting utilizes hydrogel-based composites (or biomaterial inks) that are deposited in a pattern, forming a substrate onto ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...