This work was supported by NIH grants: HL671795, HL048743, and HL108630.

1. Preparation of SDF-1&#945; Glycan Nanoparticles
Owing to the purpose of in vivo delivery, sterilize all containers, pipettes, and tips used in the preparation.
<ol>
	<li>Prepare the following stock solutions in UltraPure Water: 1% dextran sulfate; 1 M NaOH (sterile filtered with a PES membrane); 0.1% chitosan in 0.2% glacial acetic acid (filter through 0.8 and 0.22 &#956;m filters consecutively and adjust pH to 5.5 with NaOH afterward); 0.1 M ZnSO4; 15% mannitol; and 0.92 mg/ml SDF-1&#945; (stored in aliquots at 80 &#176;C, and kept at 4 &#176;C once thawed).</li>
	<li>Sterilize stock solutions through 0.22 &#956;m filter membranes. Assess endotoxin levels in the prepared solution with limulus amebocyte lysate (LAL) gel clot assay. Ensure that the levels are &#60;0.06 EU/ml.</li>
	<li>Add 0.18 ml UltraPure water to a 1.5 ml glass vial containing a magnetic stir bar. Set the stir speed at 800 rpm. Add 0.1 ml 1% dextran sulfate and stir for 2 min. Add 0.08 mg SDF-1&#945; (0.087 ml of 0.92 mg/ml SDF-1&#945; solution) and stir for 20 min.</li>
	<li>Add 0.33 ml 0.1% chitosan dropwise and stir for 5 min. Change stir speed to the maximum and add 0.1 ml 0.1 M ZnSO4 slowly with a 0.1 ml syringe (over 1 min).</li>
	<li>Return stir speed to 800 rpm and stir for 30 min. Add 0.4 ml 15% mannitol and stir for 5 min. Transfer the reaction mixture to a 1.5 ml microfuge tube. Centrifuge at 16,000 x g at 4 &#176;C for 10 min. 
	Note: The presence of mannitol facilitates the resuspension of the particles after centrifugation. Depending on the compactness of the pellet, mannitol concentration can be varied from 0% to 5%. Complete resuspension of the particles after each centrifugation is critical to avoid aggregates in the final suspension.</li>
	<li>Aspirate supernatant and use a pipette to remove the last drop of liquid slowly. Add 0.2 ml 5% mannitol. Suspend the pellet with a 0.5 ml, 29 G needle syringe. Add 1 ml 5% mannitol. Centrifuge at 16,000 x g for 15 min.</li>
	<li>Repeat step 1.6.</li>
	<li>Aspirate the supernatant. Resuspend the pellet in 0.2 ml 5% mannitol. Store the particle suspension at 4&#160;&#176;C. 
	Note: The particle suspension can also be stored frozen at -80 &#176;C or lyophilized. Including 5% mannitol in the suspension is essential to prevent aggregation of the particles after freezing and thawing or after lyophilization and rehydration. Mannitol can be removed by centrifugation of the suspension after the storage.</li>
</ol>
2. Measurement of Particle Size and Zeta Potential
The particle size and zeta potential are analyzed by dynamic light scattering and electrophoretic light scattering, respectively, with a particle analyzer indicated in Material List.
<ol>
	<li>Set up the following parameters for particle size measurement: Accumulation times: 70; Repeat times: 4; Temperature: 25 &#176;C; Diluent: water; Intensity adjustment: auto.</li>
	<li>Dilute the SDF-1&#945;-DS-CS sample with water (10-fold dilution). Load 100 &#956;l of the sample to a disposable UV cuvette such as an Eppendorf cuvette. Insert the cuvette into the cell holder. Wait for the intensity adjustment to reach &#8220;Optimum&#8221; (~10,000 cps). Start data acquisition.</li>
	<li>After the measurement is completed, record the cumulants results of diameter (nm) and polydispersity index. Average the results obtained from each of the 4 repeated readings and calculate the standard deviation.</li>
	<li>Load 500 &#956;l of the 10-fold diluted particle sample to a flow cell for zeta potential measurement. Set up the following parameters for the measurement: Accumulation times: 10; Repeat times: 5; Temperature: 25 &#176;C; Diluent: water; Intensity adjustment: auto. Record the results of the zeta potential (mV). Average the result obtained from each of the 5 repeated readings, and calculate the standard deviation.</li>
</ol>
3. Quantification of SDF-1&#945; in the Particles
<ol>
	<li>Dilute free form SDF-1&#945; to concentrations of 0.01, 0.02, 0.03, 0.04, and 0.05 mg/ml in 1.3x Laemmli sample buffer. Dilute 6 &#181;l SDF-1&#945;-DS-CS samples with 40 &#181;l 1.3x Laemmli sample buffer. (Prepare 4x Laemmli stock buffer containing 0.25 M Tris-HCl, pH 7.5, 8% SDS, 40% glycerol, 0.05 mg/ml bromophenol blue, and 8% 2-mercaptoethanol. Divide the stock solution into small aliquots and keep at -20 &#176;C for single use.)</li>
	<li>Heat the samples at 100 &#176;C for 10 min. Vortex the samples twice (each for 10 sec at maximum speed) during the 10 min heating time in order to dissociate the particles completely. Cool down the samples to RT for 2 min. Centrifuge at 10,000 x g for 0.5&#8211;1 min to bring down the water condensate. Vortex again to mix well. 
	Note: At this point, the sample solution should be clear with no visible precipitate present.</li>
	<li>Load 10 &#181;l sample/well to a 4&#8211;20% SDS gel. Run electrophoresis at 200 V for 20 min. Stain the gel with Coomassie blue protein stain.</li>
	<li>Examine the protein band density of SDF-1&#945; using a molecular imager and densitometry analysis software. Calculate the quantity of SDF-1&#945; in the particles against a standard curve constructed with free SDF-1&#945; standards.</li>
</ol>
4. In Vitro Release Assay
<ol>
	<li>Mix SDF-1&#945; glycan particles with Dulbecco&#8217;s phosphate buffered saline without calcium and magnesium (D-PBS) at a 1:1 ratio (v/v).</li>
	<li>Divide the particle suspension into 0.05 ml aliquots in 1.5 ml tubes. Load the samples to the bottom of the tubes. Avoid introducing air bubbles or disturbing the surface of the liquid. Seal the top of the tubes with Parafilm. 
	NOTE: In doing so, the liquid will remain at the bottom during the subsequent top-to-bottom rotation on the tube mixer.</li>
	<li>Rotate the tubes at 37 &#176;C on a Rotating Micro-Tube Mixer. Remove aliquots from the tubes at the designated times and immediately centrifuge the samples at 16,000 x g for 10 min at 4 &#176;C.</li>
	<li>Separate the supernatants and pellets, and reconstitute the pellet with 0.05 ml D-PBS. Store the samples at &#8722;20 &#176;C. After all the samples are collected, examine the supernatants and pellets on a SDS gel as described above.</li>
</ol>
5. Migration Assay
This assay measures the chemotactic activity of SDF-1&#945;. Interaction of SDF-1&#945; with its receptor (CXCR4) on the cell surface causes migration of the cell towards the SDF-1&#945; gradient. In this assay, the cells are loaded into an upper well (separated by a semipermeable membrane from a lower well) and SDF-1&#945; solution into a lower well.
<ol>
	<li>Dilute SDF-1&#945; (free or particle-bound form) with migration buffer (RPMI-1640 medium containing 0.5% bovine serum albumin) in a 3-fold serial dilution to final concentrations of 100, 33, 11, 3.7, 1.2, 0.41, 0.14, and 0.05 ng/ml.</li>
	<li>Add 0.6 ml of the diluted SDF-1&#945; solution or migration buffer only (negative control) to a well in the 24-well plate. Add 0.57 ml migration buffer to a well (input cell control). Incubate at 37 &#176;C for 30 min. Place a permeable cell culture insert such as Transwell (pore size 5 &#956;m, diameter 6.5 mm) on top of the lower well. Load 0.1 ml Jurkat cells (5 &#215; 105) into the Transwell insert. Load 0.03 ml cells directly to the input cell control well. Incubate the plate at 37 &#176;C for 2 hr in a 5% CO2 incubator.</li>
	<li>Remove the Transwell inserts. Transfer the cells that have migrated to the lower wells to a 4 ml polystyrene tube. Count the migrated cells with a flow cytometer.</li>
	<li>Calculate migration as a percentage of the input cell number (cell number in the input cell control well x 100/30) after subtraction of the numbers in negative controls (cells migrated to the wells containing only migration buffer).</li>
</ol>

<ol>
	<li>Delair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloidal+polyelectrolyte+complexes+of+chitosan+and+dextran+sulfate+towards+versatile+nanocarriers+of+bioactive+molecules.">Colloidal polyelectrolyte complexes of chitosan and dextran sulfate towards versatile nanocarriers of bioactive molecules.</a> Eur J Pharm Biopharm. 78 (1), 10-18 (2011).</li><li>Morachis, J. M., Mahmoud, E. A., Almutairi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+strategies+for+therapeutic+delivery+by+using+polymeric+nanoparticles.">Physical and chemical strategies for therapeutic delivery by using polymeric nanoparticles.</a> Pharmacol Rev. 64 (3), 505-519 (2012).</li><li>Yue, Z. G., 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=Surface+charge+affects+cellular+uptake+and+intracellular+trafficking+of+chitosan-based+nanoparticles.">Surface charge affects cellular uptake and intracellular trafficking of chitosan-based nanoparticles.</a> Biomacromolecules. 12 (7), 2440-2446 (2011).</li><li>Ghadge, S. K., Muhlstedt, S., Ozcelik, C., Bader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDF-1alpha+as+a+therapeutic+stem+cell+homing+factor+in+myocardial+infarction.">SDF-1alpha as a therapeutic stem cell homing factor in myocardial infarction.</a> Pharmacol Ther. 129 (1), 97-108 (2011).</li><li>Sharma, M., Afrin, F., Satija, N., Tripathi, R. P., Gangenahalli, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-derived+factor-1/CXCR4+signaling:+indispensable+role+in+homing+and+engraftment+of+hematopoietic+stem+cells+in+bone+marrow.">Stromal-derived factor-1/CXCR4 signaling: indispensable role in homing and engraftment of hematopoietic stem cells in bone marrow.</a> Stem Cells Dev. 20 (6), 933-946 (2011).</li><li>Sadir, R., Baleux, F., Grosdidier, A., Imberty, A., Lortat-Jacob, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+stromal+cell-derived+factor-1alpha-heparin+complex.">Characterization of the stromal cell-derived factor-1alpha-heparin complex.</a> J Biol Chem. 276 (11), 8288-8296 (2001).</li><li>Amara, 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=Stromal+cell-derived+factor-1alpha+associates+with+heparan+sulfates+through+the+first+beta-strand+of+the+chemokine.">Stromal cell-derived factor-1alpha associates with heparan sulfates through the first beta-strand of the chemokine.</a> J Biol Chem. 274 (34), 23916-23925 (1999).</li><li>Sadir, R., Imberty, A., Baleux, F., Lortat-Jacob, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparan+sulfate/heparin+oligosaccharides+protect+stromal+cell-derived+factor-1+(SDF-1)/CXCL12+against+proteolysis+induced+by+CD26/dipeptidyl+peptidase+IV.">Heparan sulfate/heparin oligosaccharides protect stromal cell-derived factor-1 (SDF-1)/CXCL12 against proteolysis induced by CD26/dipeptidyl peptidase IV.</a> J Biol Chem. 279 (42), 43854-43860 (1074).</li><li>Yin, 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=SDF-1alpha+in+glycan+nanoparticles+exhibits+full+activity+and+reduces+pulmonary+hypertension+in+rats.">SDF-1alpha in glycan nanoparticles exhibits full activity and reduces pulmonary hypertension in rats.</a> Biomacromolecules. 14 (11), 4009-4020 (2013).</li><li>Huang, M., Vitharana, S. N., Peek, L. J., Coop, T., Berkland, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte+complexes+stabilize+and+controllably+release+vascular+endothelial+growth+factor.">Polyelectrolyte complexes stabilize and controllably release vascular endothelial growth factor.</a> Biomacromolecules. 8 (5), 1607-1614 (2007).</li><li>McCall, R. L., Sirianni, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLGA+nanoparticles+formed+by+single-+or+double-emulsion+with+vitamin+E-TPGS.">PLGA nanoparticles formed by single- or double-emulsion with vitamin E-TPGS.</a> J Vis Exp. (82), 51015 (2013).</li><li>Carrillo-Conde, B. R., Roychoudhury, R., Chavez-Santoscoy, A. V., Narasimhan, B., Pohl, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+synthesis+of+carbohydrates+and+functionalization+of+polyanhydride+nanoparticles.">High-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles.</a> J Vis Exp. (65), 3967 (2012).</li><li>Xu, J., Amiji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+gene+delivery+and+transfection+in+human+pancreatic+cancer+cells+using+epidermal+growth+factor+receptor-targeted+gelatin+nanoparticles.">Therapeutic gene delivery and transfection in human pancreatic cancer cells using epidermal growth factor receptor-targeted gelatin nanoparticles.</a> J Vis Exp. (59), e3612 (2012).</li><li>Lauten, E. H., 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=Nanoglycan+complex+formulation+extends+VEGF+retention+time+in+the+lung.">Nanoglycan complex formulation extends VEGF retention time in the lung.</a> Biomacromolecules. 11 (7), 1863-1872 (2010).</li><li>Schatz, C., Domard, A., Viton, C., Pichot, C., Delair, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+and+efficient+formation+of+colloids+of+biopolymer-based+polyelectrolyte+complexes.">Versatile and efficient formation of colloids of biopolymer-based polyelectrolyte complexes.</a> Biomacromolecules. 5 (5), 1882-1892 (2004).</li></ol>

The authors declare that they have no competing financial interests.

As mentioned above, the DS-CS nanoparticles are formed through charge neutralization between polyanion (DS) and polycation (CS) molecules. Since the charge interaction occurs readily during the molecular collision, the concentration of the polymer solutions and the stirring speed during mixing is critical for the size of the resultant particles. A general trend is that more diluted DS and CS solutions 15 and higher stirring speed result in smaller particles.
The formulation of the S...

Dextran sulfate (DS) and chitosan (CS) are polysaccharides with multiple substituted negatively charged sulfate groups (in DS), or positively charged amine groups (deacetylated CS). When mixed in an aqueous solution, the two polysaccharides form polyelectrolyte complexes through electrostatic interactions. The resulting complexes may form large aggregates that will be phase-separated from the aqueous solution (precipitates), or small particles that are water dispersible (colloids). The specific conditions that contribute to these outcomes have been extensively studied, and have been summarized and illustrated in detail in a recent review 1. Among these conditions, two basic requirements for producing water dispersible particles are the oppositely charged polymers must 1) have significantly different molar mass; and 2) be mixed in a non-stoichiometric ratio. These conditions will allow the charge-neutral complexed polymeric segments generated by charge neutralization to segregate and form the core of the particle, and the excess polymer to form the outer shell 1. The glycan particles described in this protocol are intended for pulmonary delivery, and are designed to be net negatively charged, and of nanometer dimensions. The negative surface charge reduces the likelihood of cellular uptake of the particles 2,3. Particles of nanometer dimension facilitate the passage through the distal airways. To achieve this goal, the amount of DS used in this preparation is in excess of CS (weight ratio 3:1); and high-molecular-weight DS (weight-average MW 500,000) and low-molecular-weight CS (MW range 50&#8211;190 kDa, 75&#8211;85% deacetylated) are used.
SDF-1&#945; is a stem cell homing factor, which exerts the homing function through its chemotactic activity. SDF-1&#945; plays an important role in homing and maintenance of hematopoietic stem cells in the bone marrow, and in recruitment of progenitor cells to the peripheral tissue for injury repair 4,5. SDF-1&#945; has a heparin-binding site in its protein sequence, which allows the protein to bind to heparin/heparan sulfate, form dimers, be protected from protease (CD26/DPPIV) inactivation, and interact with target cells via the cell surface receptors 6-8. DS has similar structural properties as heparin/heparan sulfate; thus, the binding of SDF-1&#945; to DS would be similar to that of its natural polymeric ligands.
In the following protocol, we describe the preparation of SDF-1&#945;-DS-CS nanoparticles. The procedures represent one of the formulations that have been previously studied 9. The protocol is originally adapted from an investigation of VEGF-DS-CS nanoparticles 10. A small scale preparation is described, which can be easily scaled up with the same stock solutions and preparation conditions. After preparation, the particles are characterized by examining their size, zeta potential, extent of SDF-1&#945; incorporation, in vitro release time, and activity of the incorporated SDF-1&#945;.

<table border="0" cellpadding="0" cellspacing="0" width="489">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Name</td>
			<td style="width:136px;">Company</td>
			<td style="width:99px;">Catalog number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran sulfate</td>
			<td>Fisher</td>
			<td>BP1585-100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, low molecular weight&#160;</td>
			<td>Sigma</td>
			<td>448869</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zinc sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>204986</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Mannitol</td>
			<td>Sigma</td>
			<td>M9546</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraPure water&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>10977-023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDF-1&#945;</td>
			<td>Prepared according to reference 8.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe filter, PES membrane 0.22 um.&#160;&#160;&#160;</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic Micro Stirring Bars (2 x 7 mm)</td>
			<td>Fisher&#160;</td>
			<td>14-513-63</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass vial Kit; SUN-SRi</td>
			<td>Fisher&#160;</td>
			<td>14-823-182</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Delsa Nano C Particle Analyzer&#160;</td>
			<td>Backman Coulter</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf UVette Cuvets</td>
			<td>Eppendorf</td>
			<td>952010069</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4&#8211;20% Mini-PROTEAN TGX Gel</td>
			<td>Bio-Rad</td>
			<td>456-1096</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GelCode Blue Safe Protein Stain</td>
			<td>Fisher&#160;</td>
			<td>PI-24592</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Imager VersaDoc MP 4000 System</td>
			<td>BioRad</td>
			<td>170-8640</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning Transwell Permeable Supports</td>
			<td>Corning</td>
			<td>3421</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accuri C6 Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>D8537</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pyrogent plus kit</td>
			<td>Fisher</td>
			<td>NC9753738</td>
		</tr>
	</tbody>
</table>

preparation and characterization of sdf-1&#945;-chitosan-dextran sulfate nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The size and zeta potential of the prepared SDF-1&#945;-DS-CS particles are determined with a particle analyzer. Figure 1 shows the analysis of the size measurement. From the cumulants results obtained from four repeated measurements, the average hydrodynamic diameter of the SDF-1&#945;-DS-CS particles is 661 &#177; 8.2 (nm) and the polydispersity is 0.23 &#177; 0.02. The result of the zeta potential measurement is shown in Figure 2. From the five repeated measurements, the zeta potentia...

Watch this Scientific Journal Video about Preparation and Characterization of SDF-1Î±-Chitosan-Dextran Sulfate Nanoparticles at JoVE.com

Preparation and Characterization of SDF-1&amp;#945;-Chitosan-Dextran Sulfate Nanoparticles

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

preparation-characterization-sdf-1-chitosan-dextran-sulfate

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Chemistry

12.3K Views.  Brigham and Women's Hospital and Harvard Medical School. The objective of this protocol is to incorporate SDF-1α, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1α from the nanoparticles.

Video: Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, 1,4-benzene-bis(iminoguanidinium) (BBIG), is demonstrated. The ligand is synthesized as the chloride salt (BBIG-Cl) by in situ imine condensation of terephthalaldehyde with aminoguanidinium chloride in water, followed by crystallization as the sulfate salt (BBIG-SO4). Alternatively, BBIG-Cl is synthesized ex situ in larger scale from ethanol. The sulfate separation ability of the BBIG ligand is demonstrated by selective and quantitative crystallization of sulfate from seawater. The ligand can be recycled by neutralization of BBIG-SO4 with aqueous NaOH and crystallization of the neutral bis-iminoguanidine, which can be converted back into BBIG-Cl with aqueous HCl and reused in another separation cycle. Finally, 35S-labeled sulfate and &#946; liquid scintillation counting are employed for monitoring the sulfate concentration in solution. Overall, this protocol will instruct the user in the necessary skills to synthesize a ligand, employ it in the selective crystallization of sulfate from aqueous solutions, and quantify the separation efficiency.

A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...