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

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. 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…

Representative Results

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…

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…

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