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>
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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 ...

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