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).

Manyetit Gel 1. Sentezi <ol><li> Etil alkol ve ardından deiyonize su, ardından konsantre edilmiş hidroklorik asit kullanarak, tüm cam yıkayın. Tercihen bir kurutma fırınında, O / N kurumaya bırakın. DİKKAT! hidroklorik asit zararlı - Davlumbaz kişisel koruyucu donanım ve iş giyim; Etil alkol zararlıdır - kişisel koruyucu ekipman giyin. </li><li> De gaz kadar deiyonize H2O 500 ml yavaşça 30 dakika boyunca N2 gazı geçirilmesi yolu ile, bir Dreschel şişe kullanın. </li><li> Set-up Kimyasal bir davlumbaz içinde manyetit sentez cihazı. <ol><li> Bir isomantle ısıtıcı içinde 500 ml&#39;lik bir üç boyunlu yuvarlak tabanlı bir şişeye yerleştirilir ve bir kelepçe ile merkezi boyun güvence altına almak ve dur. </li><li> Yuvarlak dipli bir reaksiyon yan boyunlarının birinin içine bir kauçuk septum ve diğer yan boyun bir lastik septum ile bir geri akış kondansatörü takın. Sürekli reflü Kondansatörden soğuk su çalıştırın. </li><li> Delinme incie yuvarlak tabanlı bir şişe lastik bir N2 gaz hattına bağlı olan bir iğne ile septum ve kabarcık çalışan bir gaz hattına bağlı bir iğne ile geri akış kondenseri lastik septum, delinme (yani, su ile bir şişeye), gaz çıkışı görselleştirmek için. </li><li> Bir yandan çarklı adaptör aracılığıyla yuvarlak dipli merkezi boynuna bıçak raket yükleyin. Bir stand üzerine monte edilen bir havai karıştırıcı bıçak pedini mili takın. </li></ol></li><li> N 2 gazı ile yuvarlak dipli temizleyin ve düşük ama algılanabilir hızda akan N2 gazı bırakın. </li><li> Yuvarlak dipli bir şişede ila geri akış kondansatörü çıkarın ve demir (III) klorür, demir 0,6125 g (II) klorid tetrahidrat 1.000 g ekleyin ve gazı alınmış H2O 50 mi DİKKAT! demir (III) klorür ve demir (II) klorür tetrahydrate zararlı - kişisel koruyucu ekipman giyin. </li><li> 50 ısıtılırken, geri akış kondansatörü yerine yerleştirin ve 1000 rpm&#39;de karıştırma° C. Bu koşullar altında karıştırılarak 10 nm çapında manyetit nanopartıküllerin üretir. </li><li> Bir kez 50 ° C &#39;de, yine karıştırılırken yuvarlak dipli bir şişede lastik septum ile enjekte edilerek% 28 amonyum hidroksit çözeltisi 10 ml. DİKKAT! amonyum hidroksit zararlıdır - kişisel koruyucu ekipman giyin. Not: amonyum hidroksit çözeltisi manyetit çökeltilmesi için kullanılır ve çözelti, siyah dönmelidir. </li><li> Hala karıştırılarak amonyak gazı kaynatmak 90 ° C&#39;ye yuvarlak dipli bir şişeye ve ısı kauçuk septum ve N2 gazı hattı çıkarın. Not: Bu, geri akış kondenseri lastik septum batırılarak yuvarlak tabanlı bir şişeye N2 akışı sağlamak için isteğe bağlı olarak, ancak, maghemit için manyetit oksidasyonu Bu adım sırasında ihmal edilebilir düzeydedir. </li><li> Karıştırmaya devam ederken bir kez 90 ° C &#39;de, yuvarlak dipli bir şişeye oleik asit 1 ml. Oleik asit Na kaplamak için manyetit kullanılannoparticles manyetit jel oluşturmak için. DİKKAT! oleik asit zararlıdır - kişisel koruyucu ekipman giyin. </li><li> Yuvarlak dipli bir reaksiyon üzerine kauçuk septum ve N2 gazı hattı değiştirin ve geri akış kondansatörü çıkarın. </li><li> 2 saat boyunca 500 rpm&#39;de ısı ve karıştırın kapatın. </li><li> Isomantle ısıtıcı yuvarlak dipli çıkarın ve manyetit jel korumak için şişenin dibine düzenlenen güçlü bir mıknatıs kullanırken kalan herhangi bir sıvı süzün. DİKKAT! hasar veya yaralanmayı önlemek için son derece dikkatli güçlü bir mıknatıs anlaştım. </li><li> (Isteğe bağlı) / N hava kuru O kadar manyetit jel izin verin. </li></ol> Manyetit Gel 2. saflaştırılması <ol><li> Manyetit jel erimesi için yuvarlak dipli bir şişeye heksan 40 ml ilave edilir DİKKAT! hekzan zararlı - Davlumbaz kişisel koruyucu donanım ve çalışmalarını giyerler. </li><li> Kalıntı H2O ileri geri çıkarmak için gazı alınmış, H2O, 40 ml ile bir ayırma hunisi kullanarakmanyetit çözümü m. <ol><li> Yavaşça bir ayırma hunisine olan H üzerine manyetit çözeltisi 2 O dökün ve yavaşça 5 dakika için iki fazlı sıvı girdap. </li><li> Dışarı boşaltın ve alt sulu kısmını atın. </li><li> Yavaş yavaş magnetit çözeltisi altına, yerleşir, böylece, ayrı bir huniye gazı alınmış H2O 40 ml ekleyin ve yavaşça girdap ve daha önce olduğu gibi süzün. </li><li> Üçüncü kez yıkamak için tekrarlayın. </li></ol></li><li> Bir Erlenmeyer şişesine manyetit çözeltisini susuz sodyum sülfat değer birkaç spatula ekleyin ve girdaplı magnetit çözeltisinden kalan herhangi bir kalıntı H2O çıkarmak için kullanılır. </li><li> Sodyum sülfat ve kalan H2O çıkarmak için bir filtre hunisi içinde 1 um&#39;lik filtre kağıdı içinden manyetit çözüm Filtre NOT: Vakum yardımı tavsiye edilir. </li><li> 50 ml&#39;lik bir buharlaştırıcı balon manyetit çözeltisini ve için heksan buharlaştırılması için bir buharlaştırıcı kullanımı, dönerAşağıdaki koşullar ile 2 h: orta derecede dönüş hızı, vakum, 50 ° C su banyosu içinde balon buharlaştırılması, uygulamalı ve 24 ° C su yoğunlaştırıcı içinde dolaşan. Not: İsteğe bağlı olarak, PLGA ile kaplandıktan önce manyetit jel saklayın. </li></ol> PLGA Shell ile Manyetit Nanopartiküller 3. Kaplama <ol><li> % 1.5 (kütle / hacim) çözelti oluşturmak için etil asetat 240 ml PLGA, 3.60 g (75/25 karışım) içinde çözülür. DİKKAT: etil asetat zararlı - Davlumbaz kişisel koruyucu donanım ve çalışmalarını giyerler. </li><li> Bir% 5.0 (kütle / hacim) çözelti oluşturmak için, bir manyetik karıştırıcı kullanılarak gazı alınmış, H 2 O 500 ml, Pluronic F-127 25,00 g çözündürülür. Not: Pluronic F-127, bir biyo-uyumlu yüzey aktif madde olarak hareket eden bir non-iyonik amfifilik blok kopolimerdir. Bu aşama 3.3.2 yağ içinde su emülsiyonu stabilize etmek yardımcı olur. </li><li> Bir microspatula kullanarak, ağırlık, cam şişeler içinde altı 0.040 g alikotları manyetit jel toplandı. PerfHer bir kısım için, aşağıdaki kaplama ve yıkama işlemi kapatýnýz. NOT: tümbölenler 4. adımda bozulma önce dondurarak kurutma en aza indirirken saflık ve verim maksimize edecek etkin kullanım ve manyetik dekantasyon sağlamak için gereklidir. <ol><li> 10 dakika boyunca ultrasonik temizleyici bir 0.040 g magnetit jel kısım ve bir plastik kapta sonikasyon PLGA solüsyonu 40 ml ekleyin. </li><li> Plastik behere Pluronic çözeltisi 80 ml ilave edilir ve 7 dakika bir yağ-içinde-su emülsiyonu olarak manyetit nano partiküller PLGA kaplama oluşturmak için hemen en yüksek ayarda laboratuar mikseri ile emülsifiye. </li><li> Hemen etil asetat buharlaştırılması için kimyasal bir çeker ocak içinde 1 saat süre ile deiyonize H2O ve sonikasyon 1 L spion seyreltilir. </li><li> Spion çözüm yanındaki güçlü bir mıknatıs yerleştirin ve yavaşça mıknatıs kahverengi SPIONs toplamak için karıştırın. Not: Bu, aralıklı birçok saat için karışmaya gerekebilirçözelti önce s SPIONs çoğu tahsil edildiğini belirten beyazımsı döner. </li><li> Mıknatıslı beherde SPIONs koruyarak sulu bir çözelti boşaltacaktır. </li><li> Şöyle SPIONs üç kez yıkayın. <ol><li> Deiyonize H2O 1 L SPIONs Askıya </li><li> 20 dakika spion çözeltisi sonikasyon. </li><li> Spion çözüm yanındaki güçlü bir mıknatıs yerleştirin ve yavaşça mıknatıs kahverengi SPIONs toplamak için karıştırın. Bu çözelti, SPIONs en toplanmıştır belirten açık döner önce aralıklı birkaç saat karışmaya gerekli olabilir. </li><li> Mıknatıslı beherde SPIONs koruyarak sulu bir çözelti boşaltacaktır. </li></ol></li></ol></li><li> Bir sulu süspansiyon halinde tek bir ağırlıklı bir cam şişe içinde, altı magnetit jel tümbölenini sentezlenebilir SPIONs toplayın. Gerektiği gibi, isteğe bağlı olarak manyetik aşırı su süzün. </li></ol> 4. FreezeSPIONs arasında KURUTMA FIRINI <ol><li> Spion çözeltisi dondurun. </li><li> -Freeze kurutmak bir liyofilizerde spion çözüm O / N. </li><li> Dondurularak kurutulmuş SPIONs tartılır. Hücre etiketlenmesi için kullanılana kadar dondurularak kurutulmuş SPIONs -20 ° C&#39;de saklanabilir. NOT: -20 ° C&#39;yi dramatik Depolama bozulması kinetik azaltır ve raf ömrünü uzatır. </li></ol> SPIONs sahip Hücreleri 5. Etiketleme <ol><li> 40 mg / ml arasında bir konsantrasyonda, fosfat tamponlu salin (PBS) içinde SPIONs bir kısım askıya alma ve 30 dakika boyunca ultrasonik titremeye maruz bırakılır. </li><li> Hücre kültürü ortamı, 5 ul / ml arasında bir konsantrasyonda bir hücre, yaklaşık konfluent şişeye spion solüsyonu ekleyin. Yavaşça şişeyi sallanan hatta dağılımını sağlamak. </li><li> 37 ° C&#39;de 16 saat boyunca inkübe hücreleri. </li><li> Yavaşça kültür ortamı aspire ve PBS ile iki kez hücreleri yıkayın. </li><li> Manyetik etiketli hücreleri toplayın ve deneyler için kullanın. </li><li> Kullanılmayan spion Çözelti 4 ° C&#39;de saklanabilir ve bize olmalıdırBir kaç ay içinde ed. Her kullanımdan önce 30 dakika sonikasyon. </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.

Herhangi bir nanoparçacık sentezi protokolü gibi, reaktif kimyasal saflığı en az bir sitotoksik etkiye sahip olacaktır, yüksek kaliteli SPIONs elde edilmesi için çok önemlidir. Oleik asit (% ≥99), demir (II) klorid tetrahidrat (≥99.99%), demir (III) klorür (≥99.99%), etil asetat da dahil olmak üzere oldukça saf reaktifler satın almak için önemlidir (HPLC derece,% ≥99.9 ), heksan (HPLC derece, ≥97.0%), amonyum hidroksit (% ≥99.99) ve sodyum sülfat (≥99.0%). Nispeten pahalı olabilir çok s...

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

Hücre ve terapötik maddelerin hedefli verilmesi hedef dışı sitelerine zararlı etkilerini en aza indirirken hedef yerde tedavi edici etkiyi konsantre biyomedikal uygulamalarda geniş bir fayda sağlayacaktır. Manyetik hücre hedefleme, verimli, güvenli ve basit dağıtım tekniğidir. Süperparamanyetik demir oksit nanopartiküller (spion) biyolojik olarak uyumlu, biyolojik olarak parçalanabilir ve manyetik alanlara duyarlı hale getirmek için hücrelere endocytosed edilebilir. Sentez işlemi manyetit (Fe 3 O 4) oluşturulmasını içerir bir poli (laktik-ko-glikolik asit) (PLGA) bir kaplama oluşturmak üzere yüksek hızlı bir emülsiyon ardından nanopartiküller. PLGA-manyetit SPIONs yaklaşık 10 nm çapında manyetit çekirdek içerir, çapı yaklaşık 120 nm olan. Kültür ortamı içinde yerleştirildiğinde, SPIONs doğal hücreler tarafından endositoze ve sitoplazmik endozomlara içindeki küçük kümeler olarak depolanır. Bu parçacıklar, hücreler yeterli manyetik kütle vermekManyetik alanlar içinde hedefleme için izin vermek. Sayısız hücre sıralama ve hedefleme uygulamaları manyetik alanlara duyarlı çeşitli hücre tiplerini render tarafından etkindir. SPIONs da tümör tedavisi veya doku lehimleme yerel hipertermi bir tıbbi görüntüleme kontrast maddesi olarak kullanılmak hedeflenen ilaç ya da gen iletimi, teşhis deneyleri, kuşağın dahil olmak üzere diğer biyomedikal uygulama çeşitliliği bulunmaktadır.

Manyetit nanopartiküller 50 ° C ve 1000 rpm&#39;de (Şekil 1) demir (III) klorür ve demir (II) klorid tetrahidrat bir sulu çözelti karıştırıldıktan bir sonucu olarak çap olarak yaklaşık 10 nm. Bu sonuçlar, magnetit nanopartiküllerinin başarılı bir sentez göstermektedir. İlk kez sentezi çalışırken toplu küçük bir örnek alınır manyetit nanopartiküllerin büyüklüğü ve şekli kontrol etmek önemlidir. Transmisyon elektron mikroskopi (TEM) bu parçacıkların görselleştir...

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.

Hücre Etiketleme ve Süperparamanyetik Demir Oksit Nanopartiküller ile Hedefleme

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 Görüntüleme.  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.

Video: 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.

Teşhis ve Tedavi İzleme Nanomalzemelerin Kayıtlı bio

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

Biyomühendislik

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.

Kök Hücreler Ferumoxytol, FDA Onaylı Demir Oksit Nanopartikül Etiketleme

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

Tıp

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.

Demir Oksit hESC ve hMSCs Etiketleme MR Görüntüleme in vivo Takip Non-Invasive Nanopartiküller

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

Biyoloji

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.

Fareler ve Merkezi Sinir Sisteminde Kök Hücre İmplantasyonu Multimodal Görüntüleme

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

Nörobilim

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.

Hücrelerin ultra hızlı Mıknatıslanma için katyonize Magnetoferritin sentezi

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>Murin Glioblastomunda Görüntü Kılavuzluğunda Tedavi Uygulaması için Demir Oksit Nanopartikülleri

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>Yazar Spotlight: Glioblastoma Tedavisi için Demir Oksit Nanopartikülleri ile Yenilikçi Kanser Tedavileri

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.

Bir Glioblastoma Multiforme Hayvan Modelinde Bir Oligonükleotid Yükünün Nanopartikül Teslimi

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 ...

Kanser Araştırmaları

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).

Hücresel Kuruluş ve Daha Sonra Kimyasal Synthetized Demir Oksit Nanopartiküllerinin Biyobozunurluğunu İzlemek için ManyetometriNin Kullanılması

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.

Manyetik Nanomalzemelerin Biyofonksiyonelleştirilmesi

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

Hücre Etiketleme ve Süperparamanyetik Demir Oksit Nanopartiküller ile Hedefleme

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Hücrelerin ultra hızlı Mıknatıslanma için katyonize Magnetoferritin sentezi

Bir Glioblastoma Multiforme Hayvan Modelinde Bir Oligonükleotid Yükünün Nanopartikül Teslimi

Bir Glioblastoma Multiforme Hayvan Modelinde Bir Oligonükleotid Yükünün Nanopartikül Teslimi

Bir Glioblastoma Multiforme Hayvan Modelinde Bir Oligonükleotid Yükünün Nanopartikül Teslimi

Hücresel Kuruluş ve Daha Sonra Kimyasal Synthetized Demir Oksit Nanopartiküllerinin Biyobozunurluğunu İzlemek için ManyetometriNin Kullanılması

Manyetik Nanomalzemelerin Biyofonksiyonelleştirilmesi