Name: preparation and characterization of sdf-1α-chitosan-dextran sulfate nanoparticles
Uploaded: 2015-01-22T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Preparation and Characterization of SDF-1ÃŽÂ±-Chitosan-Dextran Sulfate Nanoparticles at JoVE.com

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>

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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 SDF-1&#945; glycan nanoparticles can be varied. For example, the amounts and ratios of SDF-1&#945;/DS/CS used in this protocol are 0.08/0.33/1 (mg/mg/mg), respectively. Depending on the intended use of SDF-1&#945; nanoparticles, these ratios can change. If the amount of SDF-1&#945; added to the mixture is 0.04 mg (0.04/0.33/1, mg/mg/mg) instead of 0.08 mg, the particles will be smaller, ~650 nm, and the SDF-1&#945; entrapment efficiency slightly greater, 70&#8211;80% 9. However, when delivered in vivo, a greater amount of the glycan matrix will be present per SDF-1&#945;. If a smaller particle size is desired, the formulation of SDF-1&#945; glycan nanoparticles can be further modified. One alternative is to use a smaller molecular weight chitosan, as demonstrated in Ref 15. It should be noted, however, that chitosan has a high affinity for endotoxin (negatively charged lipopolysaccharides); thus, a test of endotoxin level or potentially a purification is necessary for the use of any chitosan products prior to in vivo delivery. The second alternative is to load SDF-1&#945; onto pre-formed small DS-CS nanoparticles. Since the latter can be manipulated more readily in particle size and SDF-1&#945; has a high affinity for DS, loading a small amount of SDF-1&#945; to the outer shell of the DS-CS particles can result in smaller particles.
Compared with other types of nanoparticles (e.g., PLGA11, polyanhydride12, or gelatin13 nanoparticles), the DS-CS nanoparticles have particular advantages and limitations. The major advantages of the DS-CS nanoparticles are: 1) the particle preparation does not involve organic solvents or vigorous sonication. It is, therefore, suitable for protein factor incorporation, as gentle preparation reduces the likelihood of protein inactivation; 2) the matrix of the particles has a similar structural property as extracellular matrix, which makes the particles compatible with tissue environment and is less toxic and inflammatory; and 3) glycan particle-bound SDF-1&#945; mimics a natural binding relationship between the protein and the extracellular matrix, which explains the full chemotactic activity of SDF-1&#945; after being incorporated into the glycan particles. The fully active SDF-1&#945; in the particle-bound form allows it to serve as a stationary homing factor in tissue environment. The limitations of the DS-CS nanoparticles are: 1) the diameters of the particles are generally greater than the above mentioned particles; and 2) easily collapse in salt solutions, which may not be suitable for direct injection into blood circulation.
The particle-bound SDF-1&#945; is found to be minimally released from the nanoparticles after a seven-day incubation. This phenomenon is related to the high affinity of SDF-1&#945; for heparin, which also contributes to its function in vivo. Since proteins with heparin binding domains will generally have different affinities for heparin, their release rates will likely differ. For example, in a VEGF165-DS-CS nanoparticle prepared in a similar formulation, 44% of the incorporated VEGF165 was released in the first hour and 29% was released in the next 47 hr during incubation at 37 &#176;C14. Therefore, the in vitro release pattern for a specific protein needs to be individually tested.
The purpose of preparation of the SDF-1&#945;-DS-CS nanoparticles is to deliver SDF-1&#945; to the lung and establish a stem cell homing signal in the tissue. The particles can also be delivered to other tissues for the same purpose, although the specific nanoparticle format is not required for solid tissue injection. The fact that SDF-1&#945; is stabilized by DS in the nanoparticle matrix and is minimally released from the particles supports stem cell recruitment principles, and the particle is expected to be beneficial for tissue regeneration.
Since the protein-glycan nanoparticles have a similar matrix to that of cell surface glycosaminoglycans and extracellular matrix, it is possible that the particle-incorporated proteins can be exchanged into these matrixes (The negatively charged extracellular matrix molecules can compete with the binding of the positively charged protein) in vivo. This exchange can alter the protein release kinetics, and, thus, needs to be further investigated after the initial in vitro characterization.

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><tbody><tr><td>Dextran sulfate</td><td>Fisher</td><td>BP1585-100</td><td></td></tr><tr><td>Chitosan, low molecular weight&amp;nbsp;</td><td>Sigma</td><td>448869</td><td></td></tr><tr><td>Zinc sulfate heptahydrate</td><td>Sigma</td><td>204986</td><td></td></tr><tr><td>D-Mannitol</td><td>Sigma</td><td>M9546</td><td></td></tr><tr><td>UltraPure water&amp;nbsp;</td><td>Invitrogen&amp;nbsp;</td><td>10977-023</td><td></td></tr><tr><td>SDF-1&amp;alpha;</td><td>Prepared according to reference 8.</td><td></td><td></td></tr><tr><td>Syringe filter, PES membrane 0.22&amp;nbsp;&amp;#956;m</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Magnetic Micro Stirring Bars (2&amp;nbsp;x&amp;nbsp;7&amp;nbsp;mm)</td><td>Fisher&amp;nbsp;</td><td>14-513-63</td><td></td></tr><tr><td>Glass vial Kit; SUN-SRi</td><td>Fisher&amp;nbsp;</td><td>14-823-182</td><td></td></tr><tr><td>Delsa Nano C Particle Analyzer&amp;nbsp;</td><td>Backman Coulter</td><td></td><td></td></tr><tr><td>Eppendorf UVette Cuvets</td><td>Eppendorf</td><td>952010069</td><td></td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN TGX Gel</td><td>Bio-Rad</td><td>456-1096</td><td></td></tr><tr><td>GelCode Blue Safe Protein Stain</td><td>Fisher&amp;nbsp;</td><td>PI-24592</td><td></td></tr><tr><td>Molecular Imager VersaDoc MP 4000 System</td><td>BioRad</td><td>170-8640</td><td></td></tr><tr><td>Corning Transwell Permeable Supports</td><td>Corning</td><td>3421</td><td></td></tr><tr><td>Accuri C6 Flow Cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline&amp;nbsp;</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Pyrogent plus kit</td><td>Fisher</td><td>NC9753738</td><td></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 potential of the SDF-1&#945;-DS-CS particles is -24.8 &#177; 0.5 mV. As mentioned above, the presence of 5% mannitol in the SDF-1&#945;-DS-CS particle suspension is essential for prevention of particle aggregation during a freezing and thawing process. Figure 3 shows the size measurement of the particles suspended in water and frozen at -80 &#176;C. A distinct aggregation peak is visible after thawing.
The amount of SDF-1&#945; in the SDF-1&#945;-DS-CS particles is estimated by SDS gel electrophoresis. Figure 4A shows a Coomassie blue-stained SDS gel with SDF-1&#945; standards, DS-CS nanoparticle (control particles without SDF-1&#945; incorporation, Ctrl NP) and SDF-1&#945;-DS-CS nanoparticle (SDF-1&#945; NP) samples loaded. A standard curve is constructed from the density analysis of the SDF-1&#945; standard bands (Figure&#160;4B). Calculation from the standard curve indicates that the SDF-1&#945; concentration in the SDF-1&#945; NP sample is 0.03 mg/ml (average of duplicate). Since the dilution of the sample with the sample buffer is (6+40)/6, the original sample concentration is 0.23 mg/ml. As the final volume of the particle preparation is 0.2 ml, total SDF-1&#945; in the final suspension is 0.046 mg. Considering the input mass of SDF-1&#945; in the reaction mixture is 0.08 mg, the entrapment efficiency of SDF-1&#945; in the DS-CS particles is 57%. Chitosan has been previously reported to stain with Coomassie blue. The staining, however, is not significant in the gel shown in Figure 4A. It is, perhaps, related to the quantity of chitosan (particle matrix) loaded on the gel. In any case, a complete dissociation and separation of SDF-1&#945; from the particle matrix is necessary for accurately assessing the amount of the incorporated protein. This gel also shows that SDF-1&#945; is not detectably degraded by heating at 100 &#176;C for 10 min or by the vortex process, as no bands are found between the SDF-1&#945; protein band and the dye front.
The in vitro release rate of SDF-1&#945; from the nanoparticles is determined by incubation of the particles in 50% D-PBS at 37 &#176;C for 7 days. At 0 hr, 3 hr, 8 hr, 24 hr, 48 hr, and 7 days, aliquots of the samples are removed and immediately centrifuged. The released SDF-1&#945; (in the supernatant) is separated from the particle-bound SDF-1&#945; (pellet). The samples are run on a SDS gel, and the amount of SDF-1&#945; in the supernatants and pellets are determined by Coomassie staining and densitometry analysis. A typical SDS gel of the analysis is shown in Figure 5. As demonstrated, the particle bound SDF-1&#945; is minimally released after a 7-day incubation.
The activity of the particle-bound SDF-1&#945; is measured by a migration assay (chemotactic assay), and compared to that of free SDF-1&#945;. In this assay, SDF-1&#945; in the DS-CS nanoparticles is serially diluted with a migration buffer, and incubated with Jurkat cells for 2 hr. The migrated cells are counted with a flow cytometer. The data (with SDF-1&#945; concentrations of 0.05&#8211;11 ng/ml) are plotted with non-linear regression fitting, and the EC50 is calculated. As shown in Figure 6, the particle-bound SDF-1&#945; induced the same extent of migration as that of free SDF-1&#945; at&#160;all concentrations measured. The EC50 values of the free and particle-bound forms of SDF-1&#945; are 0.55 and 0.45 ng/ml, respectively.
<img alt="Figure 1" fo:content-width="3in" src="/files/ftp_upload/52323/52323fig1large.jpg" width="500" /> 
Figure 1. Analysis screen shot showing dynamic light scattering measurement of particle size. The top two panels show the plots of the autocorrelation function, G2(&#964;), and logarithm of [G2(&#964;)-1] over time. The instrument&#39;s digital signal processor (correlator) expresses the detected light intensity as a function of delay time (&#964;). The plot shows that SDF-1&#945;-DS-CS particles caused an exponential decay of the intensity between 0.2-2 msec. To obtain the average decay constant &#60;&#915;&#62;<g>, a program analyzes the Ln[G2(&#964;)-1] plot with cumulants fitting. The first order coefficient of the derived polynomial equation is assigned to &#60;&#915;&#62;<g>, which is proportional to the slope of the curve. The second order coefficient divided by the square of&#160;&#60;&#915;&#62; <g> is the polydispersity index, which describes the intensity distribution width. From &#60;&#915;&#62; <g>the average diffusion coefficient (&#60;D&#62;<d>) and particle diameter (d) are calculated as: &#915;=D&#183;q and D = kBT/3&#960;&#951;0d (Stoke-Einstein equation). The middle two panels show the fluctuation of the size reading during the 70 accumulation measurements, and the size distribution calculated with respect to intensity in the measurement. The cumulants diameter and polydispersity index are shown at the bottom of the screen. </d></g></g></g></g>
<img alt="Figure 2" fo:content-width="3in" src="/files/ftp_upload/52323/52323fig2large.jpg" width="500" /> 
Figure 2. Analysis screen shot showing electrophoretic light scattering measurement of particle zeta potential. The top panel shows a G2(&#964;) over time plot. The gradually decaying oscillating function is typically observed in charged particles that are subjected to electrophoresis and light scattering. The function reflects the frequency shift (Doppler shift) caused by the moving particles in the electrical field. A Fourier transformation of G2(&#964;) gives the power spectrum (intensity vs. frequency plot) shown in the middle panel. The center of the frequency shift peak defines the mobility of the particles, from which the zeta potential is calculated. The calculated zeta potential is shown at the bottom of the screen.
<img alt="Figure 3" fo:content-width="3in" src="/files/ftp_upload/52323/52323fig3large.jpg" width="500" /> 
Figure 3. Particle size measurement of SDF-1&#945;-DS-CS particles after freezing and thawing in water. In the absence of mannitol, the particles form aggregates after freezing and thawing. Note the formation of an additional intensity/size peak and the increase in cumulants diameter and polydispersity compared to that of Figure 1.
<img alt="Figure 4" fo:content-width="3in" src="/files/ftp_upload/52323/52323fig4large.jpg" width="400" /> 
Figure 4. Quantification of SDF-1&#945; incorporation in DS-CS nanoparticles. (A) Photograph of Coomassie-stained SDS gel loaded with free SDF-1&#945; standards, DS-CS nanoparticle (Ctrl NP), and SDF-1&#945;-DS-CS nanoparticle (SDF-1&#945; NP) samples as indicated. The SDF-1&#945; protein bands and the gel running dye front are marked. The Ctrl NP samples are not stained with Coomassie blue and the chitosan bands (wide) are vaguely visible. (B) The bands are analyzed with a densitometer, from which the standard curve is constructed with the free SDF-1&#945; standards (average of duplicate). The amount of SDF-1&#945; in the nanoparticles is calculated against the standard curve, which gives an average value of 0.3 &#181;g/well (10 &#181;l).
<img alt="Figure 5" src="/files/ftp_upload/52323/52323fig5large.jpg" /> 
Figure 5. SDS gel showing SDF-1&#945; in vitro release time course. Coomassie-stained SDS gel shows the amount of released SDF-1&#945; (in supernatants) and bound SDF-1&#945; (in pellets) after incubation of SDF-1&#945;-DS-CS nanoparticles at 37 &#176;C for various periods of time. Re-printed with permission from9.
<img alt="Figure 6" fo:content-width="3in" src="/files/ftp_upload/52323/52323fig6large.jpg" width="300" /> 
Figure 6. Dose&#8722;response curves of SDF-1&#945; (circles) and SDF-1&#945; nanoparticle (NP) (squares) in a Jurkat cell migration assay. SDF-1&#945; in both free and nanoparticle bound forms exhibited the same activity with EC50 values of 0.55 and 0.45 ng/ml, respectively. Re-printed with permission from9.

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

Preparation and Characterization of SDF-1&#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.

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

Nanyang Technological University

Research

JoVE Journal

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-1α-Chitosan-Dextran Sulfate Nanoparticles

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Bioengineering

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 ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

Cancer Research

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 &#176;C, -20 &#176;C, -80 &#176;C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Preparation and Characterization of SDF-1α-Chitosan-Dextran Sulfate Nanoparticles

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