The authors wish to acknowledge funding from the European Regional Development Fund &#8211; FNUSA-ICRC (no. CZ.1.05/ 1.1.00/ 02.0123), the American Heart Association Scientist Development Grant (AHA #06-35185N), and the National Institutes of Health (NIH #T32HL007111).

1.合成磁铁矿凝胶<ol><li>通过用浓盐酸，随后通过离子交换水接着乙醇清洗所有玻璃器皿。晾干O / N，优选在干燥炉中。 注意！盐酸是有害的 - 佩戴个人防护装备和工作在通风橱;酒精是有害的 - 佩戴个人防护装备。 </li><li>使用Dreschel瓶中脱气500ml的去离子H 2 O的通过轻轻鼓泡N 2气30分钟。 </li><li>建立的化学通风橱内的磁铁矿合成装置。 <ol><li>放置的500毫升三颈圆底烧瓶中的电热套加热器内并使用夹子固定中心颈和站立。 </li><li>安装橡胶隔片进入圆底烧瓶的侧颈之一和回流冷凝器用橡胶隔片进入的剩余侧颈部。不断通过回流冷凝器运行冷水。 </li><li>穿刺个Ë圆底烧瓶的橡胶隔片与连接到N 2气体管线针和穿刺回流冷凝器的橡胶隔片与连接到运行到鼓泡的气体线的针（即，烧瓶与水）以可视化气体流出。 </li><li>经由桨适配器安装刀片桨到圆底烧瓶的中心颈。附加叶片桨的轴，顶部搅拌器安装到一个支架上。 </li></ol></li><li>吹扫的圆底烧瓶用N 2气和离开N 2气流入以低，但检测率。 </li><li>从圆底烧瓶中去除回流冷凝器和添加1.000克铁（III），氯化0.6125克铁（Ⅱ）四水合氯化和50脱气H 2 O的溶液 注意！氯化铁和氯化亚铁四是有害的 - 穿戴个人防护装备。 </li><li>替换回流冷凝器和搅拌以1,000rpm，同时加热到50C。在这些条件下搅拌产生10纳米直径磁铁矿纳米颗粒。 </li><li>一旦在50℃下，通过在橡胶隔膜的圆底烧瓶注入，同时仍然搅拌加入10毫升28％的氢氧化铵溶液。 注意！氢氧化铵是有害的 - 佩戴个人防护装备。 注：氢氧化铵溶液用于以沉淀磁铁矿和溶液应变黑。 </li><li>取下橡胶隔膜和 N 2气体管线从圆底烧瓶中，并加热到90℃，蒸出掉氨气，同时仍然搅拌。 注意：这是可选的通过刺穿回流冷凝器的橡胶隔片保持N 2流入圆底烧瓶中，然而，磁铁矿到磁赤铁矿的氧化作用是在此步骤中可忽略不计。 </li><li>一旦达到90℃，加入1 ml的油酸向圆底烧瓶，同时仍然搅拌。油酸是用于涂覆磁铁矿呐noparticles形成磁铁矿凝胶。 注意！油酸是有害的 - 佩戴个人防护装备。 </li><li>更换橡胶隔膜和 N 2气体管线到圆底烧瓶中并取出回流冷凝器。 </li><li>熄火和搅拌在500rpm下2小时。 </li><li>从电热套加热器除去圆底烧瓶中，并在使用保持抵靠烧瓶保留磁铁矿凝胶底部强磁铁倒出任何剩余的液体。 注意！处理强力磁铁格外小心，以免损坏或人身伤害。 </li><li>允许磁铁矿凝胶风干O / N（可选）。 </li></ol> 2.净化磁铁矿凝胶<ol><li>加入40毫升己烷进圆底烧瓶以溶解磁铁矿凝胶 注意！正己烷是有害的 - 佩戴个人防护装备和工作在通风橱。 </li><li>使用分液漏斗用40ml脱气H 2 O的，以去除残留的H 2 O的往复米磁铁矿的解决方案。 <ol><li>慢慢倒入分液漏斗内的磁铁矿溶液到的 H 2 O和轻轻摇动两相液5分钟。 </li><li>排出并丢弃下部水分数。 </li><li>慢慢加入40毫升去脱气的 H 2 O至分液漏斗中使得其沉降的磁铁矿溶液下方并轻轻地旋转并如前排水。 </li><li>重复洗第三次。 </li></ol></li><li>转移磁铁矿溶液至锥形瓶中，加几铲值得无水硫酸钠，并乱舞从磁铁矿溶液中除去任何剩余的残留H 2 O的。 </li><li>过滤通过1微米的滤纸的磁铁矿溶液在过滤漏斗以除去硫酸钠和残余H 2 O 注：建议使用真空辅助。 </li><li>磁铁矿溶液转移至50毫升烧瓶中蒸发，并使用旋转蒸发器以蒸发己烷为在以下条件下2小时：中等旋转速度，施加真空，在50℃的水浴蒸发烧瓶中，并通过冷凝器24℃的循环水。 注：可选，存储涂覆PLGA之前磁铁矿凝胶。 </li></ol> 3.涂层的磁性纳米颗粒与PLGA壳牌<ol><li>溶解3.60克的PLGA（75/25共混物）于240ml乙酸乙酯中以创建一个1.5％（M / V）溶液。注意：醋酸乙酯是有害的 - 佩戴个人防护装备和工作在通风橱。 </li><li>用磁力搅拌器以创建一个5.0％（M / V）溶液溶解25.00克尼克F-127的在500毫升脱气H 2 O的。 注：尼克F-127是充当一个生物相容的表面活性剂的非离子两亲嵌段共聚物。它有助于稳定在步骤3.3.2油包水乳液。 </li><li>使用microspatula，收集磁铁矿凝胶成加权玻璃瓶中在六0.040克等分。 PERFORM下面的涂层和洗涤过程中的每个等份。 注：等份是必要的，以确保有效的处理和磁倾析，这将最大化纯度和收率，同时最小化在步骤4中降解前，冷冻干燥。 <ol><li>添加磁铁矿凝胶的0.040克等分试样和40毫升的PLGA溶液至塑料烧杯中，并声处理的在超声波清洗10分钟。 </li><li>添加80毫升的Pluronic溶液到塑料烧杯中，并立即用实验室混合器乳化在最高设定为7分钟，以在磁铁矿纳米颗粒为油包水乳液中的PLGA涂层。 </li><li>立即稀释SPION溶液在1升去离子H 2 O和超声的1小时在化学通风橱中以蒸发乙酸乙酯。 </li><li>一个强有力的磁铁旁SPION解决方案，并轻轻搅拌，收集棕SPIONs的磁铁。 注意：这可能是必要的，以间歇性地搅拌几个小时该溶液s之前变成发白，表明大多数SPIONs的已收集。 </li><li>滗水溶液，同时保留SPIONs与磁铁的烧杯中。 </li><li>如下洗SPIONs三次。 <ol><li>暂停SPIONs于1L去离子H 2 O的</li><li>超声清洗20分钟的SPION解决方案。 </li><li>一个强有力的磁铁旁SPION解决方案，并轻轻搅拌，收集棕SPIONs的磁铁。它可能需要间歇地搅拌几小时前的溶液变清，表明大多数SPIONs的已收集。 </li><li>滗水溶液，同时保留SPIONs与磁铁的烧杯中。 </li></ol></li></ol></li><li>收集来自六个磁铁矿凝胶等分的合成为一个单一的加权玻璃小瓶作为含水悬浮液的SPIONs。根据需要可选择倒出多余的水磁。 </li></ol> 4.冻结SPIONs的-drying <ol><li>冻结SPION解决方案。 </li><li>冷冻干燥SPION解决方案O / N在冻干机。 </li><li>称重冻干SPIONs。冻干SPIONs可以储存在-20℃，直到用于细胞标记。 注：储存在-20℃下显着降低降解动力学，增加的货架寿命。 </li></ol> 5.标记细胞SPIONs的<ol><li>暂停在磷酸盐缓冲盐水（PBS）SPIONs的等分试样以40mg / ml的浓度，并超声处理30分钟。 </li><li>添加SPION溶液到细胞的几乎汇合烧瓶中的细胞培养基的5微升/毫升的浓度。确保均匀分布轻摇烧瓶中。 </li><li>孵育细胞16小时，在37℃。 </li><li>轻轻吸培养基并用PBS洗涤细胞两次。 </li><li>收集磁标记的细胞和用于实验。 </li><li>未使用的SPION溶液可以储存在4℃，并应我们在短短几个月内编。超声清洗每次使用前30分钟。 </li></ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+drug+delivery+to+magnetic+implants+for+therapeutic+applications.">Targeted drug delivery to magnetic implants for therapeutic applications.</a> J Magn Magn Mater. 293 (1), 647-654 (2005).</li><li>Granot, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinically+viable+magnetic+poly(lactide-co-glycolide)+particles+for+MRI-based+cell+tracking.">Clinically viable magnetic poly(lactide-co-glycolide) particles for MRI-based cell tracking.</a> Magn Reson Med. , (2013).</li><li>Levy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+in+vivo+biotransformation+of+iron+oxide+nanoparticles.">Long term in vivo biotransformation of iron oxide nanoparticles.</a> Biomaterials. 32 (16), 3988-3999 (2011).</li><li>Mirshafiee, V., Mahmoudi, M., Lou, K., Cheng, J., Kraft, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+corona+significantly+reduces+active+targeting+yield.">Protein corona significantly reduces active targeting yield.</a> Chem Commun (Camb). 49 (25), 2557-2559 (2013).</li><li>Salvati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin-functionalized+nanoparticles+lose+their+targeting+capabilities+when+a+biomolecule+corona+adsorbs+on+the+surface.">Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface.</a> Nat Nanotechnol. 8 (2), 137-143 (2013).</li><li>Landazuri, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+targeting+of+human+mesenchymal+stem+cells+with+internalized+superparamagnetic+iron+oxide+nanoparticles.">Magnetic targeting of human mesenchymal stem cells with internalized superparamagnetic iron oxide nanoparticles.</a> Small. 9 (23), 4017-4026 (2013).</li><li>Sun, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+labeling+of+endothelial+progenitor+cells+isolated+from+peripheral+blood+with+superparamagnetic+iron+oxide+nanoparticles.">In vitro labeling of endothelial progenitor cells isolated from peripheral blood with superparamagnetic iron oxide nanoparticles.</a> Mol Med Rep. 6 (2), 282-286 (2012).</li><li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+viability+of+transplanted+beta+cells+labeled+with+a+novel+contrast+agent+-+polyvinylpyrrolidone-coated+superparamagnetic+iron+oxide+nanoparticles+by+magnetic+resonance+imaging.">Detection of viability of transplanted beta cells labeled with a novel contrast agent - polyvinylpyrrolidone-coated superparamagnetic iron oxide nanoparticles by magnetic resonance imaging.</a> Contrast Media Mol Imaging. 7 (1), 35-44 (2012).</li><li>Song, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+efficacy+of+superparamagnetic+iron+oxide+nanoparticles+to+human+neural+stem+cells:+comparison+of+ferumoxides,+monocrystalline+iron+oxide,+cross-linked+iron+oxide+(CLIO)-NH2+and+tat-CLIO.">Labeling efficacy of superparamagnetic iron oxide nanoparticles to human neural stem cells: comparison of ferumoxides, monocrystalline iron oxide, cross-linked iron oxide (CLIO)-NH2 and tat-CLIO.</a> Korean J Radiol. 8 (5), 365-371 (2007).</li></ol>

The authors declare that they have no competing financial interests.

正如任何纳米颗粒合成的协议中，反应物的化学物质的纯度是实现高质量SPIONs，将具有最小的细胞毒性作用是至关重要的。它购买很纯试剂，包括油酸（≥99％），铁（II），氯化四水合物（≥99.99％），铁（III），氯化（≥99.99％），乙酸乙酯因此重要（HPLC级，≥99.9％ ），己烷（HPLC级，≥97.0％），氢氧化铵（≥99.99％）和硫酸钠（≥99.0％）。它是特别重要的，购买很纯和高品质的PLGA，其可以?...

Targeted delivery and capture of cells to specific sites within the body is desirable for a variety of biomedical applications. Delivery of neural stem cells to the brain by MRI-guided focused ultrasound has been proposed as a possible treatment option for neurodegenerative disease, traumatic brain injury, and stroke1. Mesenchymal stem cells are being studied for their ability to deliver anti-cancer drugs to tumors due to their natural tumor-tropic properties2,3. Cardiac stem cells have been delivered to the heart as a possible treatment for myocardial infarction4,5. Vascular stents have been developed with CD34 antibodies to capture circulating progenitor cells6. While promising, these cell targeting approaches present drawbacks including lack of cell specificity, inconsistent cell retention, and off-target cell delivery.
The overall goal of the current method is to enable magnetically directed targeting of cells for a variety of cell delivery and sorting applications. Magnetic targeting allows for controlled delivery of specific cells to a specific target site with minimal off-target effects7. The magnetic fields can be generated by implanted or external devices to safely direct the movement of magnetically-labeled cells within the body8. Numerous research efforts have focused on magnetically directed targeting of stem cells to injured tissues such as the heart9-14, retina15, lung16, skin17, spinal cord18,19, bone20, liver21, and muscle22,23 in order to improve regeneration outcomes.
Magnetic targeting of cells has also been studied extensively as a means to endothelialize implantable cardiovascular devices. A uniform and complete endothelium provides a barrier between the device and circulating blood elements to mitigate thrombosis and inflammation. Endothelial cells can be delivered to the device either prior to implantation or via the vascular system following implantation. In both cases, magnetic fields are used to capture cells to the surface of the device and retain the cells when subjected to the shear stress generated by circulating blood. Magnetic vascular stents24-27 and vascular grafts28 have both been fabricated and tested for this purpose.
Magnetic cell targeting requires a strategy for labeling cells with magnetic carrier particles. These particles can be bound to the surface of cells via antibodies or ligand/receptor pairs or they can be endocytosed into the cells. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and readily endocytosed by a variety of cell types29. These particles effectively render a cell responsive to magnetic fields and are naturally degraded over time. SPIONs provide a straightforward and safe means of magnetically labeling cells in culture for a variety of magnetic targeting and sorting applications. A method for synthesizing SPIONs with a magnetite (Fe3O4) core and poly(lactic-co-glycolic acid) (PLGA) shell is provided. In addition, a method for labeling cells in culture with SPIONs is provided.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ammonium Hydroxide solution, 28% NH3 in H2O, &#8805;99.99% trace metal basis</td>
			<td>Sigma-Aldrich</td>
			<td>338818-100ML&#160;</td>
			<td>Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Dreschel bottle, 500 mL</td>
			<td>Ace Glass</td>
			<td>5516-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate, CHROMASOLVR Plus, for HPLC, 99.9%&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>650528-1L</td>
			<td>Harmful reagent - wear personal protective equipment &#38; work in fume hood</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Filter paper, 3 cm dia, grade 1</td>
			<td>Fisher</td>
			<td>09-805P</td>
			<td>For use with glass filter funnel</td>
		</tr>
		<tr>
			<td>Glass beakers, 1 L</td>
			<td>Fisher</td>
			<td>FB-101-1000</td>
			<td>For washing SPIONs</td>
		</tr>
		<tr>
			<td>Glass filter funnel, vacuum hose adapter, fits 24/40, 30 mL</td>
			<td>Fisher</td>
			<td>K954100-0344&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial caps</td>
			<td>Fisher</td>
			<td>03-391-46</td>
			<td>For use with glass vials</td>
		</tr>
		<tr>
			<td>Glass vials, 2 mL</td>
			<td>Fisher</td>
			<td>03-391-44</td>
			<td>For collecting magnetite gel &#38; SPIONs</td>
		</tr>
		<tr>
			<td>Hexane, CHROMASOLVR, for HPLC, &#8805;97.0% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>34859-1L&#160;</td>
			<td>Harmful reagent - wear personal protective equipment &#38; work in fume hood</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td>Harmful reagent - wear personal protective equipment &#38; work in fume hood</td>
		</tr>
		<tr>
			<td>Iron(II) chloride tetrahydrate, &#8805;99.99% trace metals basis&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>380024-5G</td>
			<td>Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Iron(III) chloride anhydrous, powder, &#8805;99.99% trace metals basis</td>
			<td>Sigma-Aldrich</td>
			<td>451649-1G</td>
			<td>Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Isomantle heater, 500 mL</td>
			<td>Voight Global</td>
			<td>EM0500/CEX1</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory mixer</td>
			<td>Silverson</td>
			<td>L5M-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7670520</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspatulas</td>
			<td>Fisher</td>
			<td>21-401-25A</td>
			<td>For transfering magnetite gel</td>
		</tr>
		<tr>
			<td>NdFeB magnet, 1 in x 1 in x 1 in</td>
			<td>Amazing Magnets</td>
			<td>C1000H-M</td>
			<td>Very strong magnet, handle with care</td>
		</tr>
		<tr>
			<td>Oleic acid, &#8805;99% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>O1008-5G&#160;</td>
			<td>Store in freezer; Harmful reagent - wear personal protective equipment</td>
		</tr>
		<tr>
			<td>Overhead stirrer</td>
			<td>IKA</td>
			<td>2572201</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhead stirrer clamp</td>
			<td>IKA</td>
			<td>2664000</td>
			<td>For use with overhead stirrer</td>
		</tr>
		<tr>
			<td>Overhead stirrer H-stand</td>
			<td>IKA</td>
			<td>1412000</td>
			<td>For use with overhead stirrer</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic beakers, 250 mL</td>
			<td>Fisher</td>
			<td>02-591-28</td>
			<td></td>
		</tr>
		<tr>
			<td>PLGA PURASORB PDLG (75/25 blend)</td>
			<td>Purac</td>
			<td>PDLG 7502</td>
			<td>PDLG 7502A may be used as well; Store in freezer</td>
		</tr>
		<tr>
			<td>Pluronic F-127 powder, BioReagent, suitable for cell culture</td>
			<td>Sigma-Aldrich</td>
			<td>P2443-250G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE expandable blade paddle, 8 mm dia</td>
			<td>SciQuip</td>
			<td>SP4018</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE vessel adapter, fits 24/40, 8 mm dia paddle</td>
			<td>Monmouth Scientific</td>
			<td>PTFE Vessel Adaptor A480</td>
			<td>For use with PTFE expandable blade paddle</td>
		</tr>
		<tr>
			<td>Recirculating chiller</td>
			<td>Clarkson</td>
			<td>696613</td>
			<td>For use with rotoevaporator</td>
		</tr>
		<tr>
			<td>Reflux condenser, fits 24/40, 250 mm</td>
			<td>Ace Glass</td>
			<td>5997-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotoevaporator</td>
			<td>Clarkson</td>
			<td>216949</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom flask, 50 mL, 24/40 joint</td>
			<td>Sigma-Aldrich</td>
			<td>Z414484</td>
			<td>For use with rotoevaporator</td>
		</tr>
		<tr>
			<td>Rubber septa, fits 24/40</td>
			<td>Ace Glass</td>
			<td>9096-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Separatory funnel with stopper, 250 mL</td>
			<td>Fisher</td>
			<td>10-438E</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfate ACS reagent, &#8805;99.0%, anhydrous, granular</td>
			<td>Sigma-Aldrich</td>
			<td>239313-500G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Three neck round bottom flask, angled, 24/40 joints, 500 mL</td>
			<td>Ace Glass</td>
			<td>6948-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner perforated pan</td>
			<td>Fisher</td>
			<td>15-335-20A</td>
			<td>For use with ultrasonic cleaner</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner, 2.8 L</td>
			<td>Fisher</td>
			<td>15-335-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum controller</td>
			<td>Clarkson</td>
			<td>216639</td>
			<td>For use with rotoevaporator (optional)</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Clarkson</td>
			<td>219959</td>
			<td>For use with rotoevaporator</td>
		</tr>
	</tbody>
</table>

cell labeling and targeting with superparamagnetic iron oxide nanoparticles

细胞和治疗剂的靶向递送将通过浓缩的治疗效果在目标部位，同时最小化有害的影响，以脱靶部位受益广泛的生物医学应用。磁性细胞靶向是一种高效，安全，简单的交付技术。超顺磁性氧化铁纳米颗粒（SPION）是生物可降解的，生物相容的，并且可以被内吞进入细胞，使它们响应于磁场。合成过程涉及创建磁铁矿铁 （Fe 3 O 4）纳米颗粒随后高速乳化以形成聚（乳酸-共-乙醇酸）（PLGA）的涂层。所述PLGA-磁铁矿SPIONs是大约120纳米的直径，包括约10nm直径磁铁矿核心。时，放置在培养基中，SPIONs自然被细胞内吞和细胞质内内涵体小簇存储。这些颗粒赋予足够的磁质细胞以允许磁场内定位。许多细胞分选和靶向应用是由呈现各种细胞类型响应于磁场启用。 SPIONs具有多种其它生物医学应用，以及包括用作医学成像造影剂，靶向药物或基因递送，诊断测定，并生成局部热疗的肿瘤治疗或组织焊接。

磁铁矿纳米颗粒是大约10的直径为在50℃下，以1,000rpm（图1）搅拌铁（III），氯化铁（Ⅱ）四水合氯化的水溶液的结果纳米。这些结果表明成功合成磁铁矿纳米粒子。它来验证从批次的一个小样本试图合成首次时采取磁铁矿纳米粒子的大小和形状是重要的。透射电子显微镜（TEM）是用于可视化这些粒子的首选方法。将批料应该被丢弃，合成应再次尝试，如果磁铁矿纳米颗粒不约10nm的直...

Watch this Scientific Journal Video about Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles at JoVE.com

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

cell-labeling-targeting-with-superparamagnetic-iron-oxide

Research

JoVE Journal

Bioengineering

11.9K 次观看.  Mayo Clinic. Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

视频: Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

科研

生物工程

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling molecules at low levels and damaging molecules at high levels. Accumulation of ROS in stressed plants can damage metabolites, enzymes, lipids, and DNA, causing a reduction of plant growth and yield. The ability of cerium oxide nanoparticles (nanoceria) to catalytically scavenge ROS in vivo provides a unique tool to understand and bioengineer plant abiotic stress tolerance. Here, we present a protocol to synthesize and characterize poly (acrylic) acid coated nanoceria (PNC), interface the nanoparticles with plants via leaf lamina infiltration, and monitor their distribution and ROS scavenging in vivo using confocal microscopy. Current molecular tools for manipulating ROS accumulation in plants are limited to model species and require laborious transformation methods. This protocol for in vivo ROS scavenging has the potential to be applied to wild type plants with broad leaves and leaf structure like Arabidopsis thaliana.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

阴离子氧化铈纳米粒子催化清除体内植物活性氧的研究

Reactive oxygen species (ROS) accumulation is a hallmark of plant abiotic stress response. ROS play&#160;a dual role in plants by acting as signaling ...

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various biological activities including cell proliferation, survival, migration and differentiation. Several aberrations in the FGFR signaling pathway, owing to mutations or gene amplification events, have been identified in various types of cancers. Hence, recent research has focused on developing strategies involving therapeutic targeting of FGFRs. Current FGFR inhibitors at various stages of pre-clinical and clinical development include either small molecule inhibitors of tyrosine kinases or monoclonal antibodies, with only a few peptide- based inhibitors in the pipeline. Here, we provide a protocol using phage display technology to screen small peptides as antagonists of FGFR2. Briefly, a library of phage-displayed peptides was incubated in a plate coated with FGFR2. Subsequently, unbound phage was washed off by TBST (TBS + 0.1% [v/v] Tween-20), and bound phage was eluted with 0.2 M glycine-HCl buffer (pH 2.2). The eluted phage was further amplified and used as input for the next round of biopanning. Following three rounds of biopanning, the peptide sequences of individual phage clones were identified by DNA sequencing. Finally, the screened peptides were synthesized and analyzed for affinity and biological activity.

Herein, we present a detailed protocol for screening small peptides that bind to FGFR2 using a phage display peptide library. We further analyze the affinity of the selected peptides toward FGFR2 in vitro and its ability to suppress cell proliferation.

使用噬菌体显示肽库筛选和识别针对纤维细胞生长因子受体2的小肽

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

使用磁力学监测化学合成氧化铁纳米粒子的细胞合并和后续生物降解

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...