Name: cell labeling and targeting with superparamagnetic iron oxide nanoparticles
Uploaded: 2015-10-19T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles at JoVE.com

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><tbody><tr><td>Ammonium Hydroxide solution, 28% NH&lt;sub&gt;3&lt;/sub&gt; in H&lt;sub&gt;2&lt;/sub&gt;O, &amp;ge;99.99% trace metal basis</td><td>Sigma-Aldrich</td><td>338818-100ML&amp;nbsp;</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%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>650528-1L</td><td>Harmful reagent - wear personal protective equipment &amp;amp; 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>Evaporating flask, 50 ml, 24/40 joint</td><td>Sigma-Aldrich</td><td>Z515558</td><td>For use with rotoevaporator</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&amp;nbsp;</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 &amp;amp; SPIONs</td></tr><tr><td>Hexane, CHROMASOLVR, for HPLC, &amp;ge;97.0% (GC)</td><td>Sigma-Aldrich</td><td>34859-1L&amp;nbsp;</td><td>Harmful reagent - wear personal protective equipment &amp;amp; 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 &amp;amp; work in fume hood</td></tr><tr><td>Iron(II) chloride tetrahydrate, &amp;ge;99.99% trace metals basis&amp;nbsp;</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, &amp;ge;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, &amp;ge;99% (GC)</td><td>Sigma-Aldrich</td><td>O1008-5G&amp;nbsp;</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&amp;nbsp;</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>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, &amp;ge;99.0%, anhydrous, granular</td><td>Sigma-Aldrich</td><td>239313-500G&amp;nbsp;</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

12.0K 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

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

注册生物成像，纳米材料可用于诊断和治疗监测

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

科研

生物工程

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Ferumoxytol，一个FDA批准的氧化铁纳米粒子标记干细胞

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

医学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

生物学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

神经科学

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

<meta charset="utf8"></head><body>氧化铁纳米颗粒用于小鼠胶质母细胞瘤的图像引导治疗递送

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

<meta charset="utf8"></head><body>作者聚焦:使用氧化铁纳米颗粒治疗胶质母细胞瘤的创新癌症疗法

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

癌症研究

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 ...

Biofunctionalization of Magnetic Nanomaterials

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting agents is a crucial aspect to enhance their efficacy in diagnostics and treatments while minimizing the side effects. The benefit of magnetic nanomaterials compared to non-magnetic ones is their ability to respond to magnetic fields in a contact-free manner and over large distances. This allows to guide or accumulate them, while they can also be monitored. Recently, magnetic nanowires (NWs) with unique features were developed for biomedical applications. The large magnetic moment of these NWs enables a more efficient remote control of their movement by a magnetic field. This has been utilized with great success in cancer treatment, drug delivery, cell tracing, stem cell differentiation or magnetic resonance imaging. In addition, the NW fabrication by template-assisted electrochemical deposition provides a versatile method with tight control over the NW properties. Especially iron NWs and iron-iron oxide (core-shell) NWs are suitable for biomedical applications, due to their high magnetization and low toxicity.
In this work, we provide a method to biofunctionalize iron/iron oxide NWs with specific antibodies directed against a specific cell surface marker that is overexpressed in a large number of cancer cells. Since the method utilizes the properties of the iron oxide surface, it is also applicable to superparamagnetic iron oxide nanoparticles. The NWs are first coated with 3-aminopropyl-tri-ethoxy-silane (APTES) acting as a linker, which the antibodies are covalently attached to. The APTES coating and the antibody biofunctionalization are proven by electron energy loss spectroscopy (EELS) and zeta potential measurements. In addition, the antigenicity of the antibodies on the NWs is tested by using immunoprecipitation and western blot. The specific targeting of the biofunctionalized NWs and their biocompatibility are studied by confocal microscopy and a cell viability assay.

In this work, we provide a protocol to biofunctionalize magnetic nanomaterials with antibodies for specific cell targeting. As examples, we utilize iron nanowires to target cancer cells.

磁性纳米材料的生物功能化

Magnetic nanomaterials have received great attention in different biomedical applications. Biofunctionalizing these nanomaterials with specific targeting ...

细胞标记和超顺磁性氧化铁纳米粒子靶向

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注册生物成像，纳米材料可用于诊断和治疗监测

Ferumoxytol，一个FDA批准的氧化铁纳米粒子标记干细胞

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

在中央小鼠中枢神经系统的干细胞移植的多模态成像

对细胞的超快磁化阳离子化Magnetoferritin的合成

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

多形性胶质母细胞瘤动物模型中寡核苷酸有效载荷的纳米颗粒递送

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

磁性纳米材料的生物功能化