SDF-1α-Kitosan-Dekstran Sülfat nano partiküllerin hazırlanması ve Karakterizasyonu
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH grants: HL671795, HL048743, and HL108630.
Materials
| Name | Company | Catalog number |
| 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 um. | 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 |
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