HL671795，HL048743和HL108630：这项工作是由美国国立卫生研究院资助。

1.准备SDF-1α聚糖纳米粒子由于在体内递送的目的，消毒的所有容器，移液管，并在制备中使用的提示。 <ol><li>制备下列储备溶液在超纯水：1％硫酸葡聚糖; 1 M氢氧化钠（无菌过滤用PES膜）;在0.2％的冰醋酸0.1％的壳聚糖（过滤器通过0.8和0.22微米的过滤器，并连续调节pH值至5.5，用NaOH之后）; 0.1M的硫酸锌; 15％甘露醇;和0.92毫克/毫升，SDF-1α（在80℃下以等分试样储存，并保持在4℃，一旦解冻）。 </li><li>消毒原液通过0.22微米的滤膜。与鲎变形细胞溶解物（LAL）的凝胶凝块测定中制备的溶液进行评估的内毒素水平。保证水平是&lt;0.06 EU /毫升。 </li><li>添加0.18毫升超纯水，使含有磁搅拌棒在1.5毫升的玻璃小瓶中。在800 RPM设定搅拌速度。加入0.1毫升1％硫酸葡聚糖并搅拌2分钟。添加0.08毫克，SDF-1α（0.087毫升的0.92毫克/毫升的SDF-1α溶液）并搅拌20分钟。 </li><li>添加0.33毫升0.1％脱乙酰壳多糖的溶液，并搅拌5分钟。改变搅拌速度为最大，并添加0.1毫升的0.1M 硫酸锌慢慢用0.1毫升注射器（超过1分钟）。 </li><li>返回搅拌速度800rpm，并搅拌30分钟。添加0.4毫升15％甘露糖醇，搅拌5分钟。将反应混合物转移到1.5ml微量离心管中。离心机在16000 xg离心在4℃下进行10分钟。 注意：甘露醇的存在有利于离心后，颗粒的再悬浮。根据粒料的紧凑性，甘露醇浓度可以变化从0％到5％。每次离心后的颗粒完全悬浮关键是要避免聚集在最终悬浮液中。 </li><li>吸去上清液，并使用移液管除去的液体最后一滴缓慢。中加入0.2ml的5％甘露糖醇。暂停颗粒用0.5毫升29克东东DLE注射器。加入1毫升5％甘露醇。离心在16000×g离心15分钟。 </li><li>重复步骤1.6。 </li><li>吸出上清液。重悬在0.2ml的5％甘露糖醇的颗粒。存储颗粒悬浮液在4℃下。 注意：该微粒悬浮液也可以被冷冻储存在-80℃或冻干。在悬浮液中含5％甘露糖醇是必不可少的，以防止颗粒的聚集冷冻和解冻之后，或冻干和再水化之后。甘露醇可以通过在保存后的悬浮液的离心分离除去。 </li></ol> 2.测量粒度和Zeta电位的粒径和zeta电位是通过动态光散射和电泳光散射，分别进行分析，并在物料清单指示的颗粒分析器。 <ol><li>设置为粒度测量以下参数：累积次数：70;重复次数：4;温度：25℃;稀释剂：水;强度调节：自动。 </li><li>稀释的SDF-1α-DS-CS样品用水（10倍稀释）。加载100微升样品到一次性紫外比色皿的诸如的Eppendorf试管中。插入反应杯进入细胞支架。等待强度调节，以达到“最佳”（〜万厘泊）。开始数据采集。 </li><li>测量完成后，记录直径（nm），而多分散性指数的累积量的结果。平均从各4重复读数的所获得的结果，并计算标准偏差。 </li><li>负载500微升10倍稀释的粒子试样用于zeta电位测量的流动细胞。建立起来用于测量以下参数：累积次数：10;重复次数：5;温度：25℃;稀释剂：水;强度调节：自动。记录的ζ电位（mV）的结果。平均每个重复5次读数的所获得的结果，并计算标准deviatioñ。 </li></ol> 3.量化SDF-1α的颗粒<ol><li>稀游离形式的SDF-1α为0.01，0.02，0.03，0.04，和0.05毫克/毫升在1.3倍的Laemmli样品缓冲液中的浓度。稀释6微升的SDF-1α-DS-CS样品用40微升1.3倍Laemmli样品缓冲液中。 （准备含有0.25M三盐酸，pH值7.5，8％SDS，40％甘油，0.05毫克/毫升溴酚蓝，和8％的2-巯基乙醇的Laemmli 4倍储备缓冲。除以原液成小等份，并保持在-20 °下单使用。） </li><li>加热该样品在100℃下进行10分钟。涡流在10分钟的加热时间以完全离解的颗粒两次样品（各为10秒的最大速度）。冷却样品至室温2分钟。离心机在10,000 XG为0.5-1分钟打倒冷凝水。再次涡旋拌匀。 注意：在这一点上，将样品溶液应当清楚，没有可见的沉淀物存在。 </li><li>负载10＃181;升样品/孔至4-20％SDS凝胶。运行电泳在200V进行20分钟。染色用考马斯亮蓝染色的蛋白质凝胶。 </li><li>审查的SDF-1α使用分子成像器和光密度分析软件的蛋白条带密度。计算针对与游离的SDF-1α标准建造的标准曲线的粒子的SDF-1α的量。 </li></ol> 4. 体外释放试验<ol><li>在1混合用Dulbecco磷酸盐缓冲盐水，SDF-1α聚糖颗粒不含钙和镁（D-PBS）：1的比例（体积/体积）。 </li><li>划分颗粒悬浮到0.05毫升等分在1.5ml管。加载样品到管的底部。避免引入气泡或扰乱液体的表面上。用封口膜密封的管的顶部。 注：在这样做时，液体将在管混合器随后顶部至底部在旋转过程中保持在底部。 </li><li>旋转的管子在37＃176; C对一个旋转微管混合器。从管中取出等分试样，在指定的时间，并立即离心样品在16000×g离心10分钟，在4℃。 </li><li>分离上清液和小球，并重新构成粒料与0.05毫升D-PBS。存储所述样品在-20℃。之后所有的样品被收集，如上所述检查在SDS凝胶上清液和颗粒。 </li></ol> 5.迁移实验此测定法测定的SDF-1α的趋化活性。 SDF-1α，其在细胞表面上的受体（CXCR4）的相互作用导致对SDF-1α梯度的细胞迁移的影响。在该测定中，将细胞加载到上部井和SDF-1α溶液低级阱（通过半透膜从下部井分离）成。 <ol><li>稀SDF-1α（游离或颗粒结合形式），在一个3-FO迁移缓冲液（含有0.5％牛血清白蛋白的RPMI-1640培养基）LD连续稀释至最终浓度为100，33，11，3.7，1.2，0.41，0.14，和0.05纳克/毫升。 </li><li>添加0.6毫升稀释的SDF-1α溶液或迁移缓冲液只（阴性对照）对一个井在24孔板中。添加0.57毫升迁移缓冲井（输入单元控制）。孵育在37℃下30分钟。放置一个可渗透性细胞培养插管，如上的下部井顶部Transwell小（孔径为5μm，直径6.5毫米）。负载0.1毫升Jurkat细胞（5×10 5）插入Transwell小插入。直接加载0.03毫升细胞到输入单元的控制良好。孵育板在37℃下2小时，在5％CO 2的培养箱中培养。 </li><li>取出Transwell小刀片。传送已迁移到低孔中，以一个4毫升聚苯乙烯管中的细胞。计数迁移的细胞用流式细胞仪。 </li><li>计算迁移作为输入细胞数目（在输入单元的控制以及X 100/30单元编号）中negat数字的减法后的百分比香港专业教育学院对照（细胞迁移到仅含有迁移缓冲液的孔中）。 </li></ol>

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The authors declare that they have no competing financial interests.

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?...

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。

<table border="0" cellpadding="0" cellspacing="0" width="489">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Name</td>
			<td style="width:136px;">Company</td>
			<td style="width:99px;">Catalog number</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran sulfate</td>
			<td>Fisher</td>
			<td>BP1585-100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chitosan, low molecular weight&#160;</td>
			<td>Sigma</td>
			<td>448869</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zinc sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>204986</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Mannitol</td>
			<td>Sigma</td>
			<td>M9546</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraPure water&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>10977-023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDF-1&#945;</td>
			<td>Prepared according to reference 8.</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe filter, PES membrane 0.22 um.&#160;&#160;&#160;</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic Micro Stirring Bars (2 x 7 mm)</td>
			<td>Fisher&#160;</td>
			<td>14-513-63</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass vial Kit; SUN-SRi</td>
			<td>Fisher&#160;</td>
			<td>14-823-182</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Delsa Nano C Particle Analyzer&#160;</td>
			<td>Backman Coulter</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf UVette Cuvets</td>
			<td>Eppendorf</td>
			<td>952010069</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4&#8211;20% Mini-PROTEAN TGX Gel</td>
			<td>Bio-Rad</td>
			<td>456-1096</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GelCode Blue Safe Protein Stain</td>
			<td>Fisher&#160;</td>
			<td>PI-24592</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Imager VersaDoc MP 4000 System</td>
			<td>BioRad</td>
			<td>170-8640</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Corning Transwell Permeable Supports</td>
			<td>Corning</td>
			<td>3421</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accuri C6 Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#8217;s phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>D8537</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pyrogent plus kit</td>
			<td>Fisher</td>
			<td>NC9753738</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.

所制备的SDF-1α-DS-CS粒子的大小和ζ电位均用颗粒分析仪确定的。 图1示出了尺寸测量的分析。从来自四个重复测量得到的累积的结果，所述SDF-1α-DS-CS颗粒的平均流体动力学直径是661±8.2（nm），而多分散性是0.23±0.02。 zeta电位的测量结果示于图2中 。在5次重复测量中，SDF-1α-DS-CS粒子的ζ电位为-24.8±0.5毫伏。如上所述，在SDF-1α-DS-CS粒子悬浮液的5％甘露糖醇的存在是为在一?...

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

Preparation and Characterization of SDF-1&amp;#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.

SDF-1α - 壳聚糖硫酸葡聚糖纳米粒子的制备与表征

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

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JoVE Journal

Chemistry

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

12.3K 次观看.  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.