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

Podsumowanie

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.

Streszczenie

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-1α (Stromal cell-derived factor-1α), 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-1α-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1α is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

Wprowadzenie

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 50–190 kDa, 75–85% deacetylated) are used.

SDF-1α is a stem cell homing factor, which exerts the homing function through its chemotactic activity. SDF-1α 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-1α 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-1α to DS would be similar to that of its natural polymeric ligands.

In the following protocol, we describe the preparation of SDF-1α-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-1α incorporation, in vitro release time, and activity of the incorporated SDF-1α.

Protokół

1. Preparation of SDF-1α Glycan Nanoparticles

Owing to the purpose of in vivo delivery, sterilize all containers, pipettes, and tips used in the preparation.

1. 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 μm filters consecutively and adjust pH to 5.5 with NaOH afterward); 0.1 M ZnSO₄; 15% mannitol; and 0.92 mg/ml SDF-1α (stored in aliquots at 80 °C, and kept at 4 °C once thawed).
2. Sterilize stock solutions through 0.22 μm filter membranes. Assess endotoxin levels in the prepared solution with limulus amebocyte lysate (LAL) gel clot assay. Ensure that the levels are <0.06 EU/ml.
3. 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-1α (0.087 ml of 0.92 mg/ml SDF-1α solution) and stir for 20 min.
4. 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).
5. 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 °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.
6. 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.
7. Repeat step 1.6.
8. Aspirate the supernatant. Resuspend the pellet in 0.2 ml 5% mannitol. Store the particle suspension at 4 °C.
   Note: The particle suspension can also be stored frozen at -80 °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.

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.

1. Set up the following parameters for particle size measurement: Accumulation times: 70; Repeat times: 4; Temperature: 25 °C; Diluent: water; Intensity adjustment: auto.
2. Dilute the SDF-1α-DS-CS sample with water (10-fold dilution). Load 100 μl 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 “Optimum” (~10,000 cps). Start data acquisition.
3. 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.
4. Load 500 μl 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 °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.

3. Quantification of SDF-1α in the Particles

1. Dilute free form SDF-1α to concentrations of 0.01, 0.02, 0.03, 0.04, and 0.05 mg/ml in 1.3x Laemmli sample buffer. Dilute 6 µl SDF-1α-DS-CS samples with 40 µl 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 °C for single use.)
2. Heat the samples at 100 °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 0.5–1 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.
3. Load 10 µl sample/well to a 4–20% SDS gel. Run electrophoresis at 200 V for 20 min. Stain the gel with Coomassie blue protein stain.
4. Examine the protein band density of SDF-1α using a molecular imager and densitometry analysis software. Calculate the quantity of SDF-1α in the particles against a standard curve constructed with free SDF-1α standards.

4. In Vitro Release Assay

1. Mix SDF-1α glycan particles with Dulbecco’s phosphate buffered saline without calcium and magnesium (D-PBS) at a 1:1 ratio (v/v).
2. 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.
3. Rotate the tubes at 37 °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 °C.
4. Separate the supernatants and pellets, and reconstitute the pellet with 0.05 ml D-PBS. Store the samples at −20 °C. After all the samples are collected, examine the supernatants and pellets on a SDS gel as described above.

5. Migration Assay

This assay measures the chemotactic activity of SDF-1α. Interaction of SDF-1α with its receptor (CXCR4) on the cell surface causes migration of the cell towards the SDF-1α gradient. In this assay, the cells are loaded into an upper well (separated by a semipermeable membrane from a lower well) and SDF-1α solution into a lower well.

1. Dilute SDF-1α (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.
2. Add 0.6 ml of the diluted SDF-1α 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 °C for 30 min. Place a permeable cell culture insert such as Transwell (pore size 5 μm, diameter 6.5 mm) on top of the lower well. Load 0.1 ml Jurkat cells (5 × 10⁵) into the Transwell insert. Load 0.03 ml cells directly to the input cell control well. Incubate the plate at 37 °C for 2 hr in a 5% CO₂ incubator.
3. 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.
4. 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).

Wyniki

The size and zeta potential of the prepared SDF-1α-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-1α-DS-CS particles is 661 ± 8.2 (nm) and the polydispersity is 0.23 ± 0.02. The result of the zeta potential measurement is shown in Figure 2. From the five repeated measurements, the zeta potentia...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Dextran sulfate	Fisher	BP1585-100
Chitosan, low molecular weight	Sigma	448869
Zinc sulfate heptahydrate	Sigma	204986
D-Mannitol	Sigma	M9546
UltraPure water	Invitrogen	10977-023
SDF-1α	Prepared according to reference 8.
Syringe filter, PES membrane 0.22 μm	Millipore	SLGP033RS
Magnetic Micro Stirring Bars (2 x 7 mm)	Fisher	14-513-63
Glass vial Kit; SUN-SRi	Fisher	14-823-182
Delsa Nano C Particle Analyzer	Backman Coulter
Eppendorf UVette Cuvets	Eppendorf	952010069
4–20% Mini-PROTEAN TGX Gel	Bio-Rad	456-1096
GelCode Blue Safe Protein Stain	Fisher	PI-24592
Molecular Imager VersaDoc MP 4000 System	BioRad	170-8640
Corning Transwell Permeable Supports	Corning	3421
Accuri C6 Flow Cytometer	BD Biosciences
Dulbecco’s phosphate buffered saline	Sigma	D8537
Pyrogent plus kit	Fisher	NC9753738