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 extract, 3.0 g of peptone, 25.0 g of mannitol, and 100 ml of high purity water. Autoclave this mixture at 120 &#176;C for 20 min. Once autoclaved, deposit 5 ml of the mixture in a 90 mm x 16 mm plastic Petri dish. Allow the solution to gel at 4 &#176;C and store at this temperature until further use.</li>
	</ol>
	</li>
	<li>Rehydrate G. xylinus strain preserved in freeze-dried vials by adding 1 ml of liquid culture medium and pipetting up and down, as indicated by the manufacturer&#39;s instructions.</li>
	<li>Inoculate the Petri dishes containing semisolid media with small droplets of bacterial suspension using an inoculating loop. Make sure that the inoculum covers the entire Petri dish by moving the loop in a zig zag direction from the edge to the center of the dish.</li>
	<li>Incubate the Petri dishes at 26 &#176;C for 72 hr in an incubator without CO2. Once the incubation period is complete, small white colonies are visible. If the colonies are not immediately used, store the Petri dishes at 4 &#176;C by sealing the lid with Parafilm and placing the dishes upside down. The colonies can be stored in that way for up to 6 months.</li>
	<li>Transfer 2 ml of the liquid culture medium prepared in step (1.1.1) into each well of a 24-well tissue culture plate. Take two colonies with an inoculating needle from the inoculated Petri dishes in step (1.3) and place them into the first well of the tissue culture plate. Repeat the same procedure for the remaining 23 wells.</li>
	<li>Incubate the tissue culture plate at 30 &#176;C for 7 days. This will yield a total of 24 BNC pellicles with diameter of 16 mm and a thickness of approximately 2-3 mm diameter as depicted in Figure 1. 
	Note: Do not disturb the bacterial culture at any point during incubation period, for example by shaking the plates. During the incubation period, G. xylinus extrudes glucopyranose sugar molecules to form a polymeric crystalline mesh in the air-liquid interface, which adopts the shape and size of the flask under static cultivation conditions. This polymeric matrix, known as bacterial nanocellulose (BNC), is conspicuous at end of the incubation period.</li>
	<li>Collect the BNC pellicles from the growth media and sterilize them in 200 ml of 1% NaOH solution for 1 hr at 50 &#176;C, in order to remove all traces of G. xylinus. Optionally, stir this solution at 300 rpm using a magnetic bar and a stirring plate. Discard the NaOH solution and add 200 ml of freshly prepared 1% NaOH solution. Repeat the same process once more or until the BNC pellicles in solution acquires a translucent appearance.</li>
	<li>Rinse the BNC pellicles with water three times and store them in high purity water at RT. Make sure the BNC pellicles are completely submerged in the water and are not allowed to dry at any time.</li>
	<li>Autoclave the BNC pellicles at 121 &#176;C for 20 min. 
	Note: An in vivo subcutaneous study in the rat performed by M&#228;rtson and coworkers showed non-degradation signs of the BNC after 60 weeks implantation. Indeed, BNC is degradable in nature by microbial and fungal enzymes, which are absent in mammals. On the other hand, the biodegradability of the BNC can be the result of mechanical, chemical, and biological processes that weaken the microfibril network in vivo4.</li>
</ol>
2. Synthesis of Polymer-coated Iron Oxide Nanoparticles and Its Deposition in a Bacterial Nanocellulose Membrane
<ol>
	<li>Bubble 1,000 ml of high purity water with nitrogen gas in order to remove any dissolved oxygen in the water and replace it with nitrogen.</li>
	<li>Use a three-neck round bottom flask to prepare a solution in a 2:1 molar ratio of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) diluted with the deoxygenated high purity water. For example, use 5.4 g of FeCl3&#183;6H2O and 1.98 g of FeCl2&#183;4H2O in 10 ml of deoxygenated high purity water. If this preparation turns too viscous and difficult to stir, use 0.54 g of FeCl3&#183;6H2O and 0.198 g of FeCl2&#183;4H2O in 20 ml of deoxygenated high purity water. 
	Note: Reduce the exposure time of the FeCl2&#183;4H2O to air by weighing this chemical compound as fast as possible. Once introduced in the three-neck round bottom flask, close the three-neck round bottom flask with septum stoppers until it is connected to the nitrogen gas supply and the condenser tube.</li>
	<li>Use two 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&#39;s necks.</li>
	<li>Place 1 BNC pellicle which was prepared previously in step 1.5 (15.6 mm of diameter and 2-3 mm of thickness) in the vessel with the reactants. Make sure the sample is completely submerged in the liquid.</li>
	<li>Connect the remaining neck of the vessel to a condenser tube. Additionally, use a drying tube filled with anhydrous calcium sulfate on top of the condenser tube. Run water through the condenser tube.</li>
	<li>Seal all of the glass joints with vacuum grease.</li>
	<li>Heat the solution in a silicone oil bath to 80 &#176;C using a stirring hotplate and hold this temperature until step 2.10. Use a small magnetic stir bar to mix the reactants at 350 rpm for 5 min. Make sure the BNC is appropriately impregnated with the ferrous solution and the reactants are completely dissolved. Keep stirring the mixture until the end of the experiment. 
	Note: Utilize a thermometer to verify the temperature of the silicone oil. It should be stable to 80 &#176;C.</li>
	<li>Increase the stirring velocity to 700 rpm and add (by dropping), in a time interval of 5 min, 5 ml of ammonium hydroxide (NH4OH, 14%) to the 10 ml of ferrous solution using a pipetting needle, which has been also punched in a septum stopper. After addition of the ammonium hydroxide, the color of the solution changes from yellow/orange to black.</li>
	<li>Continue stirring the solution at 80 &#176;C for another 5 min. Avoid high-speed stirrings in order to maintain the integrity of the sample. High speeds, i.e., higher than 1,000 rpm, can destroy the sample.</li>
	<li>Lower the temperature of the solution to 30 &#176;C using the temperature control bottom of the stirring hotplate and keep stirring for another 5 min. Then, turn off the hot plate. At this point, the IONP have been incorporated into the BNC mesh.</li>
	<li>Cool the mixture down to RT and separate the magnetic nanoparticles (MNP) and BNC using a strong permanent magnet (e.g., 1 Tesla). To do this, transfer the mixture to a vessel flask and then, while keeping the magnet close to the vessel, hold the MNPs and the BNC in place while decanting the supernatant. 
	Note: Be careful when handling strong magnets since they can be harmful when used incorrectly. For steps (2.12)-(2.14) and (2.16) use the deoxygenated high purity water prepared previously in (2.1) to prevent particles from oxidation.</li>
	<li>Resuspend the MNPs and BNC in 100 ml water. Gently shake the solution to remove all the MNPs that are not strongly incorporated into the BNC. Decant the supernatant again by holding the MNPs and the BNC in place using the magnet.</li>
	<li>Wash the MNPs and the BNC several times with water until the supernatant reaches neutral pH (pH~7), as measured using a colorimetric strip.</li>
	<li>Separate the magnetic-functionalized BNC or magnetic bacterial nanocellulose (MBNC) from the MNPs using tweezers and rinse the MBNC several times with water until the water runs clear.</li>
	<li>Sterilize the MBNC by exposing the MBNC O/N to UV (110-280 nm).</li>
	<li>Autoclave 500 ml of deoxygenated high purity water at 120 &#176;C for 20 min and store the MBNC in 20 ml of this water.</li>
	<li>Aseptically, immerse the sample in 1% of PEG and stir for 2 hr at RT (37 &#176;C). This procedure improves the biocompatibility and stability of the iron oxide nanoparticles deposited in the BNC, specifically those exposed at the surface5-7. The PEG coating will be distributed over the MBNC 3D network. 
	Note: Naked IONP are easily oxidized in air because of their high chemical activity8. Even though PEG is considered a non-biodegradable material, its chemical stability depends on the applied biological conditions such as water content, pH, temperature, presence of enzymes, reactive oxygen species, reactive nitrogen species, and others9.</li>
</ol>
3. Characterization of the BNC and MBNC Pellicles
<ol>
	<li>Mechanical properties
	<ol>
		<li>Perform normal loading and unloading nanoindentation test using a Berkovich indenter. The radius of Berkovich diamond indenter is 20 nm.</li>
		<li>Use fused silica and tungsten to calibrate contact area as a function of indentation depth at RT. During the test, mount the samples on the indentation using glue. The indenter approached the samples in its thickness direction.</li>
		<li>Randomly select indentation locations on samples surfaces. Keep the spacing between 2 indents between 200-300 mm.</li>
		<li>Apply the load to the samples in steps and record the corresponding displacement of the indenter. Analyze the plot of load vs depth to find the Young&#39;s modulus.</li>
		<li>Carry out the nanoindentation test of the samples in the presence of deionized water (DI water), and test by applying load rates between 0.0001 mN/sec and 0.005 mN/sec, with peak load between 0.01 mN and 0.60 mN.</li>
		<li>Use a liquid cell and keep samples under the liquids environment. This unique setup for nanomechanical characterization immersed in a fluid environment is ideal to effectively simulate the reach biomechanical functionality of BNC and MBNC membranes.</li>
	</ol>
	</li>
	<li>Structural Characterization by SEM
	<ol>
		<li>Characterize the nanocellulose fiber structure by scanning electron microscopy (SEM).</li>
		<li>Lyophilize the samples for 24 hr at -80 &#176;C. Then mount on SEM studs, sputter with Au-Pd film for 10 sec and analyze using SEM.</li>
		<li>Takes images at a magnification of 22,000X and 60,000X, with an acceleration voltage of 5 kV.</li>
	</ol>
	</li>
	<li>Magnetic domains
	<ol>
		<li>Allow the MBNC pellicles to completely dry at RT, and subsequently expose for 5 min to a permanent magnet (1 Tesla).</li>
		<li>Immediately, carry out the magnetic force measurements using a bio-AFM according to manufacturer&#39;s protocol.</li>
		<li>For each measurement, capture first the topography features and acquire the magnetic domains during a second pass. Obtain both measurements with the bio-AFM in non-contact mode.</li>
		<li>Magnetic characterization of the nanoparticles is conducted using vibrating sample magnetometer (VSM) in the physical property measurements system (PPMS) of Quantum Design, at RT&#160;(300 K), with a magnetic field in the range of -10,000 to 10,000 Oe.</li>
	</ol>
	</li>
	<li>Cytocompatibility
	<ol>
		<li>Seed human aortic smooth muscle cells (HASMC) in a 6-well tissue culture plate at a density of 1.0x103 cells/cm2 and incubate for 24 hr in the presence of the test samples: BNC and MBNC pellicles (each with a 15.6 mm of diameter).</li>
		<li>Use populations of untreated and hydrogen peroxide treated cells as negative and positive controls, respectively.</li>
		<li>Perform the Comet assay according to manufacturer&#39;s protocols and the guidelines suggested by A. Azqueta &#38; A.R. Collins10.</li>
		<li>Use the nucleic acid dye SYBR Gold in this assay to intercalate and fluorescently label the DNA contained in the electrophoresed samples according to manufacturer&#39;s protocol. 
		Note: Cells that do not undergo any DNA damage in the presence of BNC and MBNC samples, will show a fluorescent round green nucleoid, whereas DNA damaged cells will have long comets - positive samples will have nucleoids (the head of the comet) followed by tails which contain fragmented DNA material (percentage of DNA in the tail).</li>
	</ol>
	</li>
</ol>

<ol><li>Saxena, I. M., Brown, R. M. Biosynthesis of bacterial cellulose. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSaxena%2C+IM+%22Bacterial+Nanocellulose%3A+A+Sophisticated+Multifunctional+Material%22">Bacterial Nanocellulose: A Sophisticated Multifunctional Material</a>. , 1-18 (2012).</li>
<li>Chan-Park, M. B., Shen, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomimetic+control+of+vascular+smooth+muscle+cell+morphology+and+phenotype+for+functional+tissue-engineered+small-diameter+blood+vessels%5BTitle%5D)+AND+%22J.Biomed.Mater.Res.A%22%5BJournal%5D)">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/pubmed?term=((Biocellulose-based+flexible+magnetic+paper%5BTitle%5D)+AND+%22J.+Appl.+Phys%22%5BJournal%5D)">Biocellulose-based flexible magnetic paper.</a> J. Appl. Phys. 117, (2015).</li>
<li>Märtson, M., Viljanto, J., Hurme, T., Laippala, P., Saukko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Is+cellulose+sponge+degradable+or+stable+as+implantation+material%3F+An+in+vivo+subcutaneous+study+in+the+rat%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat.</a> Biomaterials. 20, 1989(1999).</li>
<li>Illésa, E., Tombácza, E., Szekeresa, M., Tótha, I., Szabób, Á, Iván, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+carboxylated+PEG-coating+on+magnetite+nanoparticles+designed+for+biomedical+applications%5BTitle%5D)+AND+%22J.+Magn.+Magn.+Mater%22%5BJournal%5D)">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/pubmed?term=((Preventing+corona+effects%3A+multiphosphonic+acid+poly%28ethylene+glycol%29+copolymers+for+stable+stealth+iron+oxide+nanoparticles%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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/pubmed?term=((Bacterial+cellulose%2Fpoly%28ethylene+glycol%29+composite%3A+characterization+and+first+evaluation+of+biocompatibility%5BTitle%5D)+AND+%22Cellulose%22%5BJournal%5D)">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/pubmed?term=((Magnetic+iron+oxide+nanoparticles%3A+synthesis+and+surface+functionalization+strategies%5BTitle%5D)+AND+%22Nanoscale+Res.+Lett%22%5BJournal%5D)">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/pubmed?term=((On+the+biodegradability+of+polyethylene+glycol%2C+polypeptoids+and+poly+%282-oxazoline%29s%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((The+essential+comet+assay%3A+a+comprehensive+guide+to+measuring+DNA+damage+and+repair%5BTitle%5D)+AND+%22Arch.+Toxicol%22%5BJournal%5D)">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/pubmed?term=((In+vivo+application+of+tissue-engineered+blood+vessels+of+bacterial+cellulose+as+small+arterial+substitutes%3A+proof+of+concept%5BTitle%5D)+AND+%22J.+Surg.+Res%22%5BJournal%5D)">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/pubmed?term=((Influence+of+cultivation+conditions+on+mechanical+and+morphological+properties+of+bacterial+cellulose+tubes%5BTitle%5D)+AND+%22Biotechnol+Bioeng%22%5BJournal%5D)">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/pubmed?term=((Microporous+bacterial+cellulose+as+a+potential+scaffold+for+bone+regeneration%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">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/pubmed?term=((A+comparative+study+on+the+mechanical+properties+of+the+umbilical+vein+and+umbilical+artery+under+uniaxial+loading%5BTitle%5D)+AND+%22Artery+Res%22%5BJournal%5D)">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/pubmed?term=((Evaluation+of+bacterial+nanocellulose-based+uniform+wound+dressing+for+large+area+skin+transplantation%5BTitle%5D)+AND+%22Mater.+Sci.+Eng.+C%22%5BJournal%5D)">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/pubmed?term=((Making+flexible+magnetic+aerogels+and+stiff+magnetic+nanopaper+using+cellulose+nanofibrils+as+templates%5BTitle%5D)+AND+%22Nature+Nanotech%22%5BJournal%5D)">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/pubmed?term=((Magnetic+force+microscopy+and+energy+loss+imaging+of+superparamagnetic+iron+oxide+nanoparticles%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Magnetic force microscopy and energy loss imaging of superparamagnetic iron oxide nanoparticles.</a> Sci. Rep. 1 (202), 1-8 (2011).</li>
</ol>

The authors have nothing to disclose.

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 fermentation process of G. xylinus12. Similarly, the porosity of the BNC can also be introduced by incorporating porogens such as paraffin wax microspheres into the fermentation process. The resulting porosity and pore interconnectivity in this case will depend on the porogen size13.
The porous network of the BNC allows them to be functionalized with nanoparticles, e.g., for drug delivery agents. In our study, we have functionalized BNC with IONP by synthesizing and growing in situ the nanoparticles into the BNC membrane, in order to implement a magnetic protocol for rapid cell recruitment and attachment in BNC-based scaffolds. Nanomechanical tests reveal that nanoscale response of BNC behaves similarly with blood vessels14 with a very low Young&#39;s modulus, EBNC = 0.0025 GPa inside the samples to 0.04 GPa at the surface. The values obtained are in the range with those observed by Fu et al.15.
The excess of IONP could easily be removed from the BNC due to the high porosity of the material. SEM photographs showed that the nanoparticles are distributed mainly in the spaces formed by fibril interlacing and dispersed along the ribbons. The concentration of the iron species used in this protocol yielded high densely packaged IONP, which brought together the BNC&#39;s ribbons. This resulted in a MBNC with larger pores than those of the unmodified BNC. Olsson et al., who used different concentrations of FeSO4/CoCl2 salts with the same volume fraction of BNC in the synthesis of cellulose nanofibril aerogels, reported a similar increase in the BNC porosity when they changed the volume fraction of the ferromagnetic cobalt ferrite nanoparticles from 0.7% to 5.7%16. This high porosity in the MBNC can be advantageous for the deposition of drugs that increase the recovery time and avoid restenosis at damaged arterial walls.
The lack of correlation between the topographical features and magnetic phase images have also been described by B. Torre et al.17, who pointed out the independence between the topography and the magnetic signals of sparse nanoparticle films. Further characterization studies need to be conducted to determine the magnetization hysteresis (M-H) loops of the MBNC via SQUID-VSM systems.
The MBNC showed low potential for toxic effects, according to the results observed in the Comet Assay, indicating that this material is biocompatible for using in contact with cells.
The most critical steps in the procedure are related to the amount of ammonium hydroxide and the speed at which it is added, as well as ensuring the complete immersion and stirring of the BNC in the solution during the reaction. The first aspect determines the size of the resulting iron oxide nanoparticles, while the second one influence how the nanoparticles are distributed across of the BNC matrix. In order to better control the size of the MNPs, a burette with a stopcock can be used to regulate the addition by dropping ammonium hydroxide into the reaction. Small pieces of BNC that can be completely submerged in the solution are advised, e.g., sizes of approximately 1.9 cm2 for a total volume of 10 ml of solution. One limitation of this technique is the inhomogeneous distribution of the IONP inside the BNC mesh.
This protocol describes a method for incorporating iron oxide nanoparticles in BNC to form a composite. Because of the biocompatibility and the physical and mechanical properties of both the BNC and the iron oxide nanoparticles, the MBNC can be used in a variety of biomedical applications such as drug delivery systems and scaffolds for cellular growth.

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><tbody><tr><td>&lt;em&gt;Glucoacetobacter xylinus&lt;/em&gt;</td><td>ATCC</td><td>700178</td><td></td></tr><tr><td>Agar</td><td>Sigma Aldrich</td><td>A1296-500G&amp;nbsp;</td><td></td></tr><tr><td>D-Mannitol Bioxtra</td><td>Sigma Aldrich</td><td>M9546-250G&amp;nbsp;</td><td></td></tr><tr><td>Yeast Extract</td><td>BD Biosciences</td><td>212750</td><td></td></tr><tr><td>Bacteriological Peptone</td><td>Sigma Aldrich</td><td>P0556</td><td></td></tr><tr><td>Sodium Hydroxide, 50% Solution In Water</td><td>Sigma Aldrich</td><td>158127-100G</td><td></td></tr><tr><td>Iron(III) Chloride Hexahydrate</td><td>Sigma Aldrich</td><td>236489-100G&amp;nbsp;</td><td></td></tr><tr><td>Ammonium Hydroxide&amp;nbsp;</td><td>Macron Fine Chemicals</td><td>6665-46</td><td></td></tr><tr><td>Poly(Ethylene Glycol), Average Mn 400</td><td>Sigma Aldrich</td><td>202398-250G&amp;nbsp;</td><td></td></tr><tr><td>Iron (II) chloride tetrahydrate</td><td>Sigma Aldrich</td><td>44939-250G</td><td></td></tr><tr><td>Disposable Petri dish</td><td>Sigma Aldrich</td><td>BR452000</td><td></td></tr><tr><td>Disposable Inoculating Loop</td><td>Fisher Scientific</td><td>22-363-604&amp;nbsp;</td><td></td></tr><tr><td>Anhydrous Calcium Sulfate</td><td>W.A. Hammond Drierite&amp;nbsp;</td><td>13001</td><td></td></tr><tr><td>High vacuum grease</td><td>Sigma Aldrich</td><td>Z273554-1EA</td><td></td></tr><tr><td>Laboratory pipetting needle with 90&amp;deg; blunt ends</td><td>Sigma Aldrich</td><td>CAD7937-12EA</td><td></td></tr><tr><td>pH test strips&amp;nbsp;&amp;nbsp;</td><td>Sigma Aldrich</td><td>P4786-100EA</td><td></td></tr><tr><td>Round-bottom three neck angle type distilling flask</td><td>Sigma-Aldrich</td><td>CLS4965250</td><td></td></tr><tr><td>Silicone oil for oil baths</td><td>Sigma-Aldrich</td><td>85409-250ML&amp;nbsp;</td><td></td></tr><tr><td>Drying Tube</td><td>Chemglass</td><td>CG-1295-01</td><td></td></tr><tr><td>Septum Stopper, Sleeve Type</td><td>Chemglass</td><td>CG-3022-98</td><td></td></tr><tr><td>Magnetic stir bar</td><td>Chemglass</td><td>CG-2001-05</td><td></td></tr><tr><td>Condenser</td><td>Chemglass</td><td>CG-1218-01</td><td></td></tr><tr><td>Temperature Controller</td><td>BriskHeat</td><td>SDC120JC-A</td><td></td></tr><tr><td>Stirring Hotplate</td><td>Fisher Scientific</td><td>11-100-49SH&amp;nbsp;</td><td></td></tr><tr><td>Comet Assay Kit</td><td>Trevigen</td><td>4250-050-K</td><td></td></tr><tr><td>SYBR Gold Nucleic Acid Gel Stain</td><td>Life Technologies</td><td>S-11494</td><td></td></tr><tr><td>bio-AFM</td><td>JPK Instruments</td><td>NanoWizard 4a BioScience AFM</td><td></td></tr><tr><td>Nanoindenter</td><td>Micro Materials Ltd</td><td>Multi-module mechanical tester&amp;nbsp;</td><td></td></tr><tr><td>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 components.
SEM images were used to resolve the microstructure, morphology, and spatial distribution of the fibers of BNC (Figure 3) and IONP distribution in functionalized BNC (Figure 4). The BNC is formed by fine ribbons (approximately 50 nm in diameter) that form open pores across the entire network without a defined pattern. The IONP are preferentially located between the pores formed by fibril interlacing, forming clusters of 100 nm or more in size. Individual IONP are also bound along the ribbons. The MBNC exhibits a less compacted fibril structure compared to the BNC, probably because IONP bring together the BNC&#39;s ribbons. Magnetic force microscope was used to reconstruct the magnetic profile at the topography of the MBNC (Figure 5A, B). Large pores of 500 nm diameter or larger are formed in the MBNC, which were not observed in untreated BNC (Figure 5A). This is in agreement with the observations in the SEM microphotographs, where the MBNC displays a more porous structure than the unmodified BNC. A magnetic force gradient with two domains of different magnetization was detected across the MBNC surface (Figure 5B), whose contrast does not correlate with the hills and valleys formed by IONP-rich regions in the MBNC topographic images (Figure 5A). High and weak intensity magnetic fields are denoted as yellow and green in Figure 5B respectively. The hysteresis loop of the nanoparticles, which is measured embedded in the bacterial nanocellulose, is shown in Figure 5 providing evidence that all the IONPs were superparamagnetic at RT, with no hysteresis.
HASMC were cultured in the presence of BNC and of MBNC to test for any detrimental effect on the viability of individual cells as a result of exposure to these foreign materials. The extent of damage in individual cells was quantified by the detection of DNA strand breaks (Figure 6). The results were compared to HASMC growing under normal culturing conditions of 37 &#176;C, 95% air, and 5% CO2 (negative control) and to HASMC with hydrogen peroxide-induced genotoxicity (100 &#181;M H2O2) for 30 min (positive control). Paired comparisons using t-test showed that the effects of the MBNC on cell viability were significantly different from those induced with hydrogen peroxide treatment on HASMC (p-value &#60; 0.001, ***).
<img alt="Figure 1" src="/files/ftp_upload/52951/52951fig1.jpg" /> 
Figure 1. Macroscopic aspects of bacterial nanocellulose. BNC pellicles have been obtained after an 11-day incubation period, which are approx. 3 mm in thickness. The incubation period depends on the requirements for the intended use. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52951/52951fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52951/52951fig2.jpg" /> 
Figure 2. Fabrication of magnetically functionalized bacterial nanocellulose. Iron oxide nanoparticles are assembled and incorporated in situ within the BNC, yielding a MBNC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52951/52951fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52951/52951fig3.jpg" /> 
Figure 3. SEM image of BNC. The BNC displays a fine network and non-aggregated ribbons with sizes of 50 nm or less. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52951/52951fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52951/52951fig4.jpg" /> 
Figure 4. SEM image of BNC-IONP pellicle. Iron oxide nanoparticles (IONP) are preferentially positioned between the interlacing ribbons. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52951/52951fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52951/52951fig5.jpg" /> 
Figure 5. AFM topography of MBNC and magnetic domain structures. (A) Surface topography of MBNC showing spots of highly packaged nanoparticles, which stand above the nanofibril structure. (B) Yellow and green domains denote two regions of different magnetization of high and weak intensity magnetic field respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52951/52951fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52951/52951fig6.jpg" /> 
Figure 6. Extent of DNA damage in HASMC after being exposure to BNC, and MBNC respectively. PosCtl denotes HASMC that underwent hydrogen peroxide treatment for comparative purposes. NegCtl denotes HASMC growing under normal culturing conditions. The detrimental effects of the MBNC on HASMC viability were significantly different from those observed in the PosCtl (p-value&#60;0.001, ***). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52951/52951fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

13.2K Views.  University of Illinois at Urbana-Champaign. 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. 

Video: Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

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 ...

Iron Nanowire Fabrication by Nano-Porous Anodized Aluminum and its Characterization

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and data storage. We demonstrate a fabrication method for iron (Fe) nanowires&#160;via electrochemical deposition into anodic&#160;alumina oxide (AAO) templates. The templates are fabricated by anodization of aluminum (Al) discs, and the pore length and diameter are controlled by changing the anodizing conditions. Pores with an average diameter of around 120 nm are created using oxalic acid as the electrolyte. Using this method, cylindrical nanowires are synthesized, which are released by dissolving the alumina using a selective chemical etchant.

In this work, we describe a&#160;protocol to fabricate iron nanowires, including the formation of the porous alumina membrane that is used as the template, electrodeposition into templates using electrolyte solution, and the release of the nanowires into the solution.

Magnetic nanowires possess unique properties that have attracted the interest of different fields of research, including basic physics, biomedicine, and ...

Chemistry

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

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

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

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

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 ...

Biofunctionalization of Magnetic Nanomaterials

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

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

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

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 ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

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