SDF-1α - 壳聚糖硫酸葡聚糖纳米粒子的制备与表征
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
硫酸葡聚糖（DS）和脱乙酰壳多糖（CS）与多个取代带负电荷的硫酸基（在DS）的多糖，或带正电荷的胺基（脱乙酰CS）。当在水溶液中混合时，两种多糖形成通过静电相互作用的聚电解质复合物。所得复合物可以形成大的聚集体将相分离的水溶液（沉淀），或小颗粒是水可分散的（胶体）。有助于这些成果的具体条件已被广泛研究，并进行了总结，并详细示出在最近的评论1。在这些条件下，用于生产水可分散的颗粒的两个基本要求是相反电荷的聚合物必须1）有显著不同摩尔质量;和2）被混合在一个非化学计量比。这些条件将允许由电荷产生的电荷中性络合聚合物链段中和，以分隔并形成颗粒的核，和过量的聚合物，以形成外壳1。在这个协议中所描述的聚糖颗粒旨在用于肺部递送，并且被设计成净负电荷，和纳米尺寸。的负的表面电荷降低了蜂窝颗粒2,3的吸收的可能性。纳米尺寸的颗粒有利于通过远端气道的通道。为了实现这一目标，DS在这一制备中所用的量是过量的CS（重量比3：1）;和高分子量的DS（重均分子量50万）和低分子量的CS（分子量范围50-190 kDa的，75-85％脱乙酰化）被使用。
SDF-1α是干细胞归巢因子，其发挥通过其趋化活性的归巢功能。 SDF-1α起着寻和维护的造血干细胞在骨髓中的重要作用，并在招募PROGE的NITOR细胞的损伤修复4,5外围组织。 SDF-1α具有肝素结合位点在其蛋白序列，其允许蛋白结合到肝素/硫酸乙酰肝素，形式二聚体，被保护免受蛋白酶（CD26 / DPPIV）失活，并通过细胞表面受体与靶细胞相互作用6-8。 DS也有类似的结构性质肝素/硫酸乙酰肝素;因此，SDF-1α对DS中的结合将是类似于其天然的聚合物配位体。
在下面的协议中，我们描述了SDF-1α-DS-CS纳米颗粒的制备。该程序表示先前已经研究了9所述的制剂中的一个。该协议最初是改编自VEGF-DS-CS纳米粒子10进行调查。一个小规模的制备进行了说明，可以很容易地按比例放大以相同储备溶液和制备条件。制备后，该颗粒的特征在于BÝ检查其尺寸，zeta电位，SDF-1α结合的程度， 在体外释放时间，以及掺入的SDF-1α活性。
Protocol
Representative Results
Discussion
如上所述，在DS-CS纳米颗粒通过电荷中和聚阴离子（DS）和聚阳离子（CS）的分子之间形成。由于电荷相互作用的分子碰撞时容易发生时，聚合物溶液和搅拌速度的混合期间的浓度是对所产生的粒子的大小是至关重要的。一个总的趋势是，越来越稀释DS和CS的解决方案15和更高的搅拌速度导致更小的粒子。
所述SDF-1α聚糖纳米颗粒的制剂可以改变。例如，SDF-1α/ DS / CS在这?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
HL671795，HL048743和HL108630：这项工作是由美国国立卫生研究院资助。
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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